Isooctadecyl isooctadecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

11 Feb - 20 Apr 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2012)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-octyldodecyl isooctadecanoate

EC Number:

298-361-4

EC Name:

2-octyldodecyl isooctadecanoate

Cas Number:

93803-87-3

IUPAC Name:

93803-87-3

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: pale yellow liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: N567701
- Storage condition of test material: at room temperature in the dark
- Other: density approximately 860 kg/m³ (25 ºC)

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured human peripheral lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: F10 complete culture medium, containing Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin.

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
48 h treatment, 48 h harvest time, without metabolic activation: 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 48 h harvest time, with metabolic activation: 1000 µg/mL

Experiment 2:
24 h treatment, 24 h harvest time, without metabolic activation: 100, 333 and 1000 µg/mL
3 h treatment, 24 h harvest time, with metabolic activation: 100, 333 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.9% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycine C (0.2 µg/mL, 24 h treatment time, -S9; 0.1 µg/mL, 48 h treatment time, -S9) and cyclophosphamide (15 µg/mL in nutrient medium, 3 h treatment time, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h with metabolic activation, 24 and 48 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h fixation time with and without metabolic activation

SPINDLE INHIBITOR (cytogenetic assays): 0.5 µg/mL colcemid, added 3 hours before harvest time (21 h with metabolic activation, 21 and 45 h without metabolic activation)
STAIN (for cytogenetic assays): 5% (v/v) Gimsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (100 per culture, duplicate cultures)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER:
Blood samples were taken from healthy, adult male volunteers by venapuncture, using a sterile vessel containing sodium heparine. The blood samples were stored at 4-25 ºC. Cultures were started within 4 h after blood collection. The average generation time of the cells is 13.6-14.1 h. Cultures were incubated at 37 ºC for 48 h prior to exposure to the test substance.

Evaluation criteria:

A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range (min = 0, max = 5 (mean = 0.9, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0, max = 5 (mean = 0.7, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded)
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
c) A homogeneous response between the replicate cultures is observed.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) A statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at 1000 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
A range-finding test was performed to select the dose levels for the main experiment, using 10, 33, 100, 333 and 1000 µg/mL, with and without metabolic activation. The test substance precipitated in the culture medium at 1000 µg/mL (see Table 1).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed at any concentration, with or without metabolic activation (see Table 2 and 3).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: results of range-finding assay

 

Metabolic activation	Test substance concentration (μg/mL)	Mitotic index (%)
		24 h harvesting time	48 h harvesting time
-	Vehicle control (DMSO)	100	100
-	10	91	98
-	33	86	98
-	100	91	105
-	333	93	105
-	1000	100	91
+	Vehicle control (DMSO)	100
+	10	92	-
+	33	90	-
+	100	94	-
+	333	98	-
+	1000	94	-

Table 2: Chromosome aberrations, summarised data, experiment 1

 

Metabolic activation	Test substance concentration (μg/mL)	Total number of metaphases analysed	Total number of aberrations^1		Number of cells with aberrations^1		Mitotic index (%)^2
			Incl. gaps	Excl. gaps	Incl. gaps	Excl. gaps
24 h treatment, 24 h harvesting time
-	Vehicle control (DMSO 1%)	200	1	1	1	1	100
-	100	200	7	6	6	5	98
-	333	200	1	1	1	1	106
-	1000	200	3	2	3	2	106
-	mitomycin C	200	41	37	38***	34***	61
48 h treatment, 48 h harvesting time
-	Vehicle control (DMSO 1%)	200	1	1	1	1	100
-	1000	200	1	1	1	1	81
-	mitomycin C	150	72	65	59***	55***	49
3 h treatment, 24 h harvesting time
+	Vehicle control (DMSO 1%)	200	4	2	4	2	100
+	100	200	4	4	4	4	68
+	333	200	0	0	0	0	81
+	1000	200	1	1	1	1	73
+	cyclophosphamide	200	89	75	71***	63***	33
3 h treatment, 48 h harvesting time
+	Vehicle control (DMSO 1%)	200	9	9	5	5	100
+	1000	200	1	1	1	1	81

¹gaps include chromatid and isochromatid (chromosome) gaps

²percentage of metaphases per 1000 cells, compared to control

* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)

 

 

 

Table 3: Chromosome aberrations, summarised data, experiment 2

 

Metabolic activation	Test substance concentration (μg/mL)	Total number of metaphases analysed	Total number of aberrations^1		Number of cells with aberrations^1		Mitotic index (%)^2
			Incl. gaps	Excl. gaps	Incl. gaps	Excl. gaps
24 h treatment, 24 h harvesting time
-	Vehicle control (DMSO 1%)	200	0	0	0	0	100
-	100	200	2	2	2	2	99
-	333	200	4	4	4	4	113
-	1000	200	1	1	1	1	97
-	mitomycin C	150	63	63	53***	53***	39
3 h treatment, 24 h harvesting time
+	Vehicle control (DMSO 1%)	200	3	3	3	3	100
+	100	200	2	2	2	2	108
+	333	200	2	2	2	2	101
+	1000	200	2	2	2	2	104
+	Cyclophosphamide	200	111	109	80***	79***	32

¹gaps include chromatid and isochromatid (chromosome) gaps

²percentage of metaphases per 1000 cells, compared to control

* P < 0.05; ** P < 0.01; *** P < 0.001 (Chi-square test)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative