Isooctadecanoic acid, 1,1'-(2,2-dimethyl-1,3-propanediyl) ester

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:

26 May - 13 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study. No historical positive control data, but positive control chemicals produced a statistically significant increase in the incidence of cells with chromosome aberrations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

(no historical positive control data), according to current OECD Guideline 473 adopted 2014, at least 300 well-spread metaphases should be scored per concentration

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

109884-54-0

Cas Number:

109884-54-0

IUPAC Name:

109884-54-0

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: clear colourless liquid
- Analytical purity: 100%
- Batch No.: OE31003999
- Expiration date of the batch: 03 October 2017
- Stability under test conditions: in vehicle ethanol: stable
- Storage condition of test material: at room temperature in the dark
- Hygroscopic: yes, store in well-sealed container
- Volatile: no

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

0.4 ml whole blood of a healthy male donor treated with heparin was added to 5 mL or 4.8 ml culture medium and 0.1 mL (9 mg/mL) Phytohaemagglutinin and was cultured for 48 h and thereafter exposed to selected doses of the test substance.
- Type and identity of media: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/ml heparin
- Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT:
Dose range finding study / First cytogenetic assay: age 22, AGT = 12.8 h
Second cytogenetic assay: age 31, AGT = 13.5 h (24 h treatment); age 35, AGT = 13.2 h (48 h treatment)
- Environmental conditions: humidity: 80 - 100% (actual range 52 - 90%); 5.0 ± 0.5% CO2 in air, temperature: 37.0 ± 1.0°C (actual range 34.5 - 37.4°C)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate, prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg)

Test concentrations with justification for top dose:

Dose range finding test:
- 1.7, 5.4, 17, 52, 164, 512, 1600 µg/mL (without S9-mix, 24 h and 48 h exposure time)
Experiment 1:
- 5.4, 17, 52 µg/mL (with and without S9-mix, 3 h exposure time)
Experiment 2:
- 17, 164, 1600 µg/mL (without S9-mix, 24 h and 48 h exposure time)

Vehicle / solvent:

- Vehicle used: ethanol (final concentration in culture medium: 0.5 - 1.0% (v/v))
The test substance was difficult to dissolve in aqueous solutions. It was soluble in ethanol at concentrations of 102.4 mg/mL and below but formed a suspension at the concentration of 160 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: Mitomycin C (0.5 and 0.75 µg/mL for 3 h, 0.2 and 0.3 µg/mL for 24 h, 0.1 and 0.15 µg/mL for 48 h exposure period); +S9-mix: Cyclophosphamide (10 µg/mL for 3 h exposure period (24 h fixation time)); Solvent: Hanks’ Balanced Salt Solution (HBSS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Dose range finding test: 24 and 48 h
First cytogenetic assay: 3 h
Second cytogenetic assay: 24 h and 48 h
- Fixation time (start of exposure up to fixation or harvest of cells):
Dose range finding test: 24 and 48 h
First cytogenetic assay: 24 h
Second cytogenetic assay: 24 h and 48 h

SPINDLE INHIBITOR: colchicine (0.5 µg/mL medium)
STAIN: 5% (v/v) Giemsa solution in Sörensenbuffer pH 6.8

NUMBER OF REPLICATIONS: Duplicates in two independent experiments

NUMBER OF CELLS EVALUATED:
100 metaphase chromosome spreads per culture were examined for chromosome aberrations. (200 metaphase chromosome spreads were examined for one solvent control since a high incidence of cells with aberrations was observed)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

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

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant 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 none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test, one-sided, p < 0.05

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(test substance was tested beyond the limit of solubility)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the culture medium at a concentration of 52 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
At the 24 h and 48 h continuous exposure time single blood cultures were treated with 1.7, 5.4, 17, 52, 164, 512 and 1600 µg test substance/mL culture medium without S9-mix. The test substance was tested beyond the limit of solubility.
The test substance was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean number of cells with chromosome aberrations found in the solvent control cultures was within or just above the laboratory historical control data range. Since clear negative results were observed in all test substance concentrations this deviation does not have any influence on study integrity.
The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.

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 first cytogenetic assay

Test item	Concentration	Mitotic index	Number of aberrant cells
	in µg/mL	in %	with gaps	without gaps
Exposure period 3 h, fixation time 24 h, without S9-mix
Ethanol	0.5% (v/v)	100	1	1
Test substance	5.4 µg/mL	98	1	0
	17 µg/mL	92	1	1
	52 µg/mL^1	95	5	4
Mitomycin C	0.5 µg/mL	66	58*	58*
Exposure time 3 h, fixation time 24 h, with S9 -mix
Ethanol	0.5% (v/v)	100	1	1
Test substance	5.4 µg/mL	99	2	1
	17 µg/mL	98	0	0
	52 µg/mL^1	96	2	1
Cyclophosphamide	10 µg/mL	50	58*	58*

¹ the test substance precipitated in the culture medium

* Significantly different from control group (Chi-square test)

Table 2: Results of second cytogenetic assay

Test item	Concentration	Mitotic index	Number of aberrant cells
	in µg/mL	in %	with gaps	without gaps
Exposure period 24 h, fixation time 24 h, without S9 -mix
Ethanol	0.5% (v/v)	100	0	0
Test substance	17 µg/mL	89	4	4
	164 µg/mL^1	63	0	0
	1600 µg/mL^1	55	0	0
Mitomycin C	0.2 µg/mL	53	52*	52*
Exposure period 48 h, fixation time 48 h, without S9 -mix
Ethanol	0.5% (v/v)	100	10^2	9^2
Test substance	17 µg/mL	92	5	5
	164 µg/mL^1	91	3	3
	1600 µg/mL^1	99	4	4
Mitomycin C	0.1 µg/mL	88	61*	60*

¹ The test substance precipitated in culture medium

² Both slides of the B culture were examined for chromosome aberrations

* Significantly different from control group (Chi-square test)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative