Icosane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no in vitro genetic toxicity data available for Icosane. However, data is available for structural analogues Hydrocarbons, C14 -C18, n-alkanes, isoalkanes, <2% aromatics, Hydrocarbons, C14-C17, n-alkanes, <2% aromatics, Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics, Isohexadecane, and Hydrodesulfurized Kerosene. This data is read across to Icosane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

All read across genetic toxicity tests listed below had negative results for Icosane.

 

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

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

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

Genetic Toxicity in vitro - Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

From December 20, 1993 to February 4, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Ames test (5 Salmonella strains), GLP. Substance identification: information available from supplier for commercial name Substance analytical certificate available

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ames test

Principles of method if other than guideline:

Guideline principles

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Reverse gene mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Stock of S. typhimurium tester strains were obtained from B. N. Ames (University of California Berkeley, USA). Master stocks are held in liquid nitrogen and were aliquots of nutrient broth cultures then stored at -80°C. See below Table 7.6.1/1.

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared in laboratory from liver of a Sprague-Dawley male rat (IFFA CREDO, France) induced by Aroclor 1254 and stored at -80 °C as aliquots. See below Table 7.6.1/2

Test concentrations with justification for top dose:

HDF 200 was tested as an emulsion in 10% Pluronic F68 aqueous solution (1/1)( v/v) in preliminary test and both main tests.
Doses: 0 (5% Pluronic F68 solution as solvent), 2, 6, 20, 60, 200 µL/plate in Pluronic F68 aqueous solution for all S. typhimurium strains (see below Table 7.6.1/3)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 10% Pluronic aqueous solution
- Justification for choice of solvent/vehicle: the test substance (oil) was insoluble in water and other vehicles (DMSO, acetone)

Untreated negative controls:

yes

Remarks:

Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of HDF 200, the S9 mix and the medium.

Negative solvent / vehicle controls:

yes

Remarks:

Aqueous Pluronic F68 solution at 5%

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below Table 7.6.1/4

Remarks:

6 plates for negative control (solvent). 3 plates for positive controls. 3 plates for controls of sterility (S9, solvent, medium).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
After range-finding test, two independent experiments were conducted in the main test by agar plate incorporation with and without S9 mix. The different controls (negative and positive controls, controls for sterility) were tested in the same conditions.

DURATION
- Preincubation period: yes- S9 activation system preincubation at 37°C for 60 min before agar plate incorporation (when negative results in the main first test in the presence of S9 activation)
- Exposure duration: 48h at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: three scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups.


DETERMINATION OF CYTOTOXICITY
- Method: other: a preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate (test substance dilutions in Pluronic F68 solution at 10% (1/1)( v/v)).


OTHER: scoring (3 measurements/plate). The mean number and standard deviation of revertants are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent control groups: the ratio between test substance revertants and solvent negative control revertants was performed at each dose-concentration

Evaluation criteria:

CRITERIA OF DECISION:
-Biological significance:
a reproducible 2-fold increase in the number of revertants (3 times in the case of TA 1535 and TA 1537 strains) compared with the vehicle controls, in any strain at any dose-level with some evidence of a dose-relationship (3 dose-concentrations) will be considered as a positive result. Reference to historical data may be taken into account in the evaluation of the data obtained.

-Statistical significance:
the test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
-Reproductibility:
Positive results should be observed in two independently tests. Positive results observed in one test without reproducibility in two tests independently conducted should not be considered as significant (Brusick ,1980). Complementary test could be performed.

ACCEPTANCE CRITERIA:- The mean of the solvent control revertants for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the study director. The positive control compounds must induce an increase in mean revertants of at least twice (3 times in the case of strains TA 1535 and 1537) the concurrent solvent controls.The test substance must be sterile at the highest concentration after agar plate incubation 48h at 37°C.

Statistics:

The test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: no

RANGE-FINDING/SCREENING STUDIES: The substance was freely soluble in the vehicle Pluronic F68 . A preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate in 10% Pluronic F68 solution (1/1) (v/v). A slight cytotoxicity (62.4% to 102.2% of the control survival) at the highest test substance was observed in all strains. Considering the slight toxicity this concentration (200 µL/plate) was used in the main test both in the absence and the presence of S9 activation system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:no

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration (µL/plate)	TA 1535		TA 1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	28	6.8	4	2.6	17	4.1	113	7.1	265	35
2**	21	4	5	0.6	13	3.2	119	10.3	263	46.8
6**	29	5.5	5	1.7	16	1.7	99	4.7	259	27
20**	24	5.5	4	1.7	16	5.1	110	6.9	251	45.4
60**	29	9.1	3	1.5	16	8.4	105	12.5	253	32.5
200**	31	5	3	0	16	3.5	116	9.3	205	16.5
Positive control***	461	37.2	543	159	340	19.7	701	88.9	1360	206.1

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration (µL/plate)	TA 1535		TA 1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	12	3.2	6	1.9	26	5	114	6	347	19.9
2**	11	2.5	7	2.9	21	3	127	5.7	355	35.1
6**	8	1.2	5	1.2	28	3.6	136	11.7	332	47.3
20**	12	3	5	2.3	24	5.6	141	1.5	331	4.6
60**	12	3.6	4	1	24	5.6	124	5.2	375	99.7
200**	9	0.6	7	3.2	29	5.6	137	8	375	19.2
Positive control***	892	104.6	94	10.5	1324	117.2	2653	361.7	1380	87

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration (µL/plate)	TA 1535		TA 1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	24	3.7	4	2.5	15	3.6	106	11.4	235	15.9
2**	28	5.3	4	2.9	15	4	113	10.6	273	33.1
6**	25	12.1	7	2.3	15	3	108	13.1	240	54.1
20**	25	4.7	4	1.2	15	6.4	108	11.7	225	28.3
60**	19	6	7	2.1	18	1.7	92	5	243	32.5
200**	21	4	5	1.2	18	4.6	105	3.2	225	31.9
Positive control***	825	67.1	133	21.4	318	29.4	1151	235.3	1213	41.6

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/8: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Second test)

Test substance concentration (µL/plate)	TA 1535		TA 1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	9	5.1	4	1.7	18	6.3	138	12.6	395	45.1
2**	9	0.6	7	4.2	26	3.6	170	23	403	47.7
6**	10	2.6	7	0.6	29	10.7	183	19.2	373	25.7
20**	10	1.7	7	1.5	34	2.5	195	7	379	12.2
60**	9	1.7	7	0	33	11.6	213	9.3	384	41.8
200**	11	1	7	0.6	37	3.5	232	12.2	385	47.4
Positive control***	127	18.6	91	6	1457	193.5	1093	142.3	953	97.9

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/9: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Third test)

Test substance concentration (µL/plate)	TA 98		TA 100
	Mean	Standard deviation	Mean	Standard deviation
0*	19	5.2	107	14.6
2**	26	2.6	100	8.5
6**	25	10.3	118	7.6
20**	31	0	153	5.7
60**	32	5.5	124	14.4
200**	30	1.5	135	17.6
Positive control***	1457	193.5	1305	51.4

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Conclusions:

Interpretation of results:
negative with and without metabolic activation

Under the test conditions, HDF 200 did not demonstrate any in vitro mutagenic activity in the Salmonella test system.

Executive summary:

The mutagenic potential of HDF 200 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 102, TA 98 and TA 100 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 48 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was tested in the main experiment according to two tests independently performed in the same way as the range-finding test. The test substance was diluted in 10% Pluronic F68 aqueous solution. Dose levels used in the main assay were 0 (solvent), 2, 6, 20, 60 and 200 µL/plate in the main test, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test (62.4% to 102.2% survival). No increase in revertant mean number was observed in any S. typhimurium strain with and without S9-mix in the preliminary test and in the first main test. However, positive results were observed in the second main test conducted with 60-minute S9 pre-incubation before plate incorporation.

Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Both statistically significant results and biologically significant results (two-fold increase by comparison with solvent) were observed at the highest test substance dose in S. typhimurium TA 98 but without a dose-effect relationship. Reduced but statistically significant positive results were also observed in S. typhimurium TA 100 with a dose-effect relationship. However, no biological significance was observed at any dose. A third test was performed in S. typhimurium TA 98 and TA 100 under the same conditions as the second main test. In this case the positive results obtained in the previous main test were not observed in either S. typhimurium TA 98 or TA 100. In the absence of reproducible results, the test substance was not considered as mutagenic in S. typhimurium according to the decision criteria of Brusick (1980).

Under the conditions of this study, HDF 200 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Reference 2

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: supporting information

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

Key result

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 CLP Regulation (1272/2008).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Equivalent or similar to OECD Guideline 476.

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 40.8 x 10^-6

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material induced a good range of toxicities for evaluation of the test material (percent relative growths, 65.3% to 2.8%). The toxicities did show some variability between replicate samples. In the presence of metabolic activation, no indication of mutagenic activity was observed. The average cloning efficencies for the solvent and untreated negative controls varied from 119.1% without activation to 82.7% with activation which demonstrated very good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive compounds yielded normal mutant frequencies that were greatly in excess of the background.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Executive summary:

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

The test material was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Further testing is not required based on the negative results obtained in read across in vitro chromosome aberration test, bacterial reverse mutation assay, mammalian cell gene mutation test and sister chromatid exchange assays conducted. Icosane is therefore not classified as a mutagen. Furthermore, there is no evidence from the read across repeat dose toxicity studies that Icosane is able to induce hyperplasia or pre-neoplastic lesions.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There is no genetic toxicity data available for Icosane. However, data is available for structural analogues Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics, Hydrocarbons, C14-C17, n-alkanes, <2% aromatics, Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics, Isohexadecane, and Hydrodesulfurized Kerosene. This data is read across to Icosane 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 gene mutation study in bacteria

Hydrocarbons, C14-C17, n-alkanes, <2% aromatics

In a supporting study (Petroquimica, 1985), the test material (Hydrocarbons, C14-C17, n-alkanes, <2% aromatics) was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98 and 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver S9 fraction for metabolic activation.The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared. Apreliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The test substance was then tested in another experiment performed in the same way as the range-finding test.No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test). Positive controls gave the expected increases in the number of revertants, with and without S-9 mix.Under the conditions of this study, Petrepar 147 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key study (TOTAL, 1994), the test material (Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics)was assessed in the Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 102, TA 98 and TA 100 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 48 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was tested in the main experiment according to two tests independently performed in the same way as the range-finding test. The test substance was diluted in 10% Pluronic F68 aqueous solution. Dose levels used in the main assay were 0 (solvent), 2, 6, 20, 60 and 200 µL/plate in the main test, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test (62.4% to 102.2% survival). No increase in revertant mean number was observed in any S. typhimurium strain with and without S9-mix in the preliminary test and in the first main test. However, positive results were observed in the second main test conducted with 60-minute S9 pre-incubation before plate incorporation.

Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Both statistically significant results and biologically significant results (two-fold increase by comparison with solvent) were observed at the highest test substance dose in S. typhimurium TA 98 but without a dose-effect relationship. Reduced but statistically significant positive results were also observed in S. typhimurium TA 100 with a dose-effect relationship. However, no biological significance was observed at any dose. A third test was performed in S. typhimurium TA 98 and TA 100 under the same conditions as the second main test. In this case the positive results obtained in the previous main test were not observed in either S. typhimurium TA 98 or TA 100. In the absence of reproducible results, the test substance was not considered as mutagenic in S. typhimurium according to the decision criteria of Brusick (1980). Under the conditions of this study, HDF 200 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

In a supporting study (Carless, 1997a), areverse gene mutation assay in bacteria was conducted according to OECD 471 in compliance with GLP. Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 were exposed to test material (Hydrocarbons, C14 -C18, n-alkanes, isoalkanes, cyclics, <2% aromatics) at concentrations of 0 (as solvent control), 0.1, 0.33, 1.0, 3.33 and 10 mg/plate in the presence and absence of S9 metabolic activation. Cytotoxicity was not observed at any dose level for all strains. Under the test conditions, Clairsol NS did not induce a revertant increase in any strain, in the presence or the absence of S9, up to the highest dose (10.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, the test material was not mutagenic in S.typhimurium in the absence and the presence of S9 activation system.

Isohexadecane

In a reverse gene mutation assay in bacteria (EC Erdolchemie, 1990) and in compliance with Good Laboratory Practice, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to Isohexadecan at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation. No cytotoxicity was observed with all the dose tested. Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used (+/- S9). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, Isohexadecan did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.

In Vitro Chromosome Aberration in Mammalian Cells

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

In a key OECD Guideline 473 study (Exxon, 1991), the potential of the test material (Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics) to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substance.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 CLP Regulation (1272/2008).

 

In vitro Gene Mutation study in Mammalian Cells

Hydrodesulfurized Kerosene

In a key study (API, 1984), exposure to eight graded doses of the test material (Hydrodesulfurized Kerosene) in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore Hydrodesulfurized Kerosene is not considered to be mutagenic in this test system.

 

In vitro Sister Chromatid Exchange Assay in Mammalian Cells

Hydrodesulfurized Kerosene

In a supporting study (API, 1987), the test material (Hydrodesulfurized Kerosene) was soluble at all concentrations tested. The study in both the presence and absence of S9 was repeated since there was a poor metaphase cell yield. The responses to the positive and negative control materials fulfilled the requirements for the assays. The test material did not cause an increase in SCEs in the absence of exogenous activation. The test material did cause a increase in SCEs at two non adjacent doses (0.05 and 0.4 uL/mL) in the activation assay. However, the increased activity was only seen in one of two treatment flasks. These increases appeared to be random and of no biological significance. It was concluded that the test material was negative in the SCE assay.

In Vivo

Further testing is not required based on the negative results obtained in read across in vitro chromosome aberration test, bacterial reverse mutation assay, mammalian cell gene mutation test and sister chromatid exchange assays conducted. Icosane is therefore not classified as a mutagen. Furthermore, there is no evidence from the read across repeat dose toxicity studies that Icosane is able to induce hyperplasia or pre-neoplastic lesions.

Justification for classification or non-classification

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