Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

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:

20 Apr - 1 Sep 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (ECHA, 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)

Version / remarks:

adopted in 1997

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

Remarks:

The department of health of the government of the United Kingdom

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

IUPAC Name:

Reaction product of pentaerythritol and trimethylolpropane with n-pentanoic acid, 2-methylbutyric acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid

Details on test material:

- Name of test material (as cited in study report): Only trade name available
- Physical state: colourless liquid
- Analytical purity: 100% (UVCB)
- Storage condition of test material: room temperature in the dark

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Eagle's minimal essential medium supplemented with sodium bicarbonate, H EPES buffer, L-glutamine, peniciIlin/streptomycin, amphotericin B and 15% foetal calf serum

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, 4h: -S9: 39.06, 78.13, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 1, 4h: +S9: 39.06, 78.13, 156.25*, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 2, 20h: -S9: 39, 78.1, 156.25, 312.5, 625*, 1250*, 2500*, 5000* µg/mL
Experiment 2, 4 h, +S9: 156.25, 312.5, 625, 1250*, 2500*, 5000* µg/mL

* dose levels selected for metaphase analysis

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Exp I and II, - S9: Ethyl methansulphonate (EMS) 750 and 500 µg/mL; Exp I and II, + S9: Cyclophosphanamide (CP) 25 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
SPINDLE INHIBITOR (cytogenetic assays): demelcocine (Colcemid) 0.1 µg/mL
STAIN (for cytogenetic assays): Giemsa 5%
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

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, but tested up to precipitating concentrations

Remarks:

Exp1: an oily precipitate at and above 625 µg/mL, +S9 and –S9; Exp. 2: oily precipitate was observed at and above 1250 µg/mL, -S9, and at and above 2500 µg/mL, +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH when the test material was dosed into media
- Effects of osmolality: there was no increase in osmolality by more than 50 mOSM

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1

The qualitative assessment of the slides determined that there were scorable metaphase up to the maximum recommended dose level of 5000 µg/mL in the without and with metabolic activation (S9) groups. An oily precipitate was observed at and above 625 µg/mL in both treatment groups.

With regard to the mitotic index data, these findings show that no toxicity was observed up to the maximum recommended dose level of 5000 µg/mL in the absence or presence of S9. No toxicity was observed at or below the precipitating dose level of 625 µg/mL. Therefore the maximum five dose levels were selected for metaphase analysis.

Both of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material induced statistically significant increases in the frequency of cells with gap-type aberration at 2500 and 5000 µg/mL in the absence of S9 mix and at 5000 µg/mL in the presence of S9 mix. There were no statistically significant increases in the frequency of cells with aberrations excluding gaps and therefore the observation were considered to have no toxicological significance.

The test material did not induce a significant increase in the numbers of polyploidy cells at any dose level in either of the treatment cases.

Experiment 2

The qualitative assessment of the slides determined, as in Experiment 1, that were scorable metaphases up to the maximum recommended dose level, 5000 µg/mL.

An oily precipitate was observed at and above 1250 µg/mL in absence of S9 mix and at and above 2500 µg/mL in the presence of S9 mix.

The data on mitotic index confirm that qualitative observations in that no dose-related inhibition of mitotic index was observed.

The precipitation of the test material appeared to have no effect on toxicity. Therefore, doses below that precipitating dose level and up to 5000 µg/mL were selected for metaphase analysis.

Both the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control treatments gave statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore satisfactory and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either including or excluding gaps, in the presence of metabolic activation (at 2% concentration) or with continuous 20 h exposure in the absence of S9 mix. Therefore, the small increases observed in Experiment 1 were confirmed to be of no toxicological significance.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases.

Tab. 1: Experiment 1: 4 h treatment, 20 h harvest - Without Metabolic Activation

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	0.0	0.0
312.5 µg/mL	105	-	-	-
625 µg/mL	96	0.5	1.5	1.0
1250 µg/mL	110	1.0	1.0	1.0
2500 µg/mL	92	0.0	2.5	0.0
5000 µg/ml	94	0.0	2.5	0.5
EMS 750 µg/mL	75	0.0	18.0	10.0

EMS: ethyl methanesulphonate; solvent control: acetone

  Tab. 2: Experiment 1: 4 h treatment, 20 h harvest- With Metabolic Activation (S9-Mix)

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.5	0.5	0.0
156.25 µg/mL	-	-	2.0	1.0
312.5 µg/mL	92	-	-	-
625 µg/mL	102	0.0	2.0	1.5
1250 µg/mL	106	0.0	1.0	0.5
2500 µg/mL	92	0.0	0.5	0.0
5000 µg/mL	101	0.0	3.5	2.0
CP 25 µg/mL	31	0.0	15.5	6.5

CP: cyclophosphamide; solvent control: acetone

 

Tab. 3: Experiment 2: 20 h treatment, 20 h harvest - Without Metabolic Activation

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	1.5	0.5
625 µg/mL	70	0.0	0.5	0.0
1250 µg/mL	81	0.0	2.5	1.5
2500 µg/mL	116	0.0	0.5	0.0
5000 µg/mL	105	-	2.5	1.5
EMS 500 µg/mL	28	0.0	51.0	44.0

EMS: ethyl methanesulphonate; solvent control: acetone

 

Tab. 4: Experiment 2: 4 h treatment, 20 h Harvest- With Metabolic Activation (S9-Mix)

Concentration	Mitotic index [%]	Polyploid cells	Aberrant cells
			incl. gaps	excl. gaps
Solvent control	100	0.0	1.0	1.0
1250 µg/mL	101	0.0	1.0	0.5
2500 µg/mL	91	0.0	0.0	0.0
5000 µg/mL	105	-	1.0	1.0
CP 25 µg/mL	27	0.5	22.0	10.0

CP: cyclophosphamide; solvent control: acetone

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative