Reaction mass of glycerol 1,3-di(acetate) and glycerol acetate and triacetin

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given. Wrong positive control at 6 h exposure without S9 mix and highest test concentration of 2.2 mg/mL.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

wrong positive control at 6 h exposure without S9, highest test concentration 2.2 mg/mL.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Triacetin

EC Number:

203-051-9

EC Name:

Triacetin

Cas Number:

102-76-1

Molecular formula:

C9H14O6

IUPAC Name:

propane-1,2,3-triyl triacetate

Details on test material:

- Name of test material (as cited in study report): Glycerol triacetate, Triacetin
- Analytical purity: 98.2 %

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Without metabolic activation
Continuous treatment: 0.55, 1.1 and 2.2 mg/mL
Short-term treatment: 0.55, 1.1 and 2.2 mg/mL

With metabolic activation
Short-term treatment: 0.55, 1.1 and 2.2 mg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 5 µg/mL, +S9; mitomycin C, 0.5 µg/mL, -S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: For continuous treatment, cells were treated for 24 or 48 h without S9.   
- Exposure duration: For short-term treatment, cells were treated for 6 h with and without S9 and cultivated with fresh media for 18 h.

NUMBER OF REPLICATIONS: 2 independent experiments were performed.

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: Cell confluency was measured with MonocellaterTM

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Statistics:

Fisher's exact analysis.

Results and discussion

Test results

Test results 1

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

other: wrong positive control at 6 h exposure, valid at 24 and 48 h exposure

Test results 2

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

Effects at highest concentration were only observed at unphysiological pH value.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2.2 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Structural chromosomal aberrations (including gaps) were induced on short-term treatment with an exogenous metabolic activation system (2.2 mg/mL). However, at this highest test substance concentration the medium colour was changed to yellow indicating a significant decrease of pH, accompanied by a high increase of cytotoxicity. These confounding factors do not allow an interpretation of the observed chromosomal damages as test-substance specific. It is known that acidification of the cell culture medium leads to chromosomal damages together with cytotoxicity in cell cultures. This is not expected to occur at similar test substance concentrations in vivo.
Polyploidy was not induced under any conditions.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Chromosome analysis of Chinese hamster lung cells (CHL/IU) continuously treated with glycerol triacetate (TA) ** without S9 mix. 

Group	Concen- tration (mg/mL)	Exposure (h)	No. of cells ana-lyzed	Cyto- toxicity^3 (%)	Mito- tic index^4 (%)	Aberrant cells in %		Poly- ploid^2 (%)
						incl. gaps	excl. gaps
Non-treatment			200	-	-	0.0	0.0	0.25
Solvent^1	0	24	200	100	-	0.5	0.5	0.5
Triacetin	0.55	24	200	100	-	1.0	1.0	0.0
Triacetin	1.1	24	200	92.5	-	1.5	1.5	0.25
Triacetin	2.2	24	200	90.5	8.6	0.0	0.0	0.0
MMC	0.00005	24	200	-	-	16.5*	15.5*	0.13
Solvent^1	0	48	200	100	-	0.0	0.0	0.25
Triacetin	0.55	48	200	105	-	0.0	0.0	0.38
Triacetin	1.1	48	200	104	-	0.0	0.0	0.38
Triacetin	2.2	48	200	85	6.8	0.0	0.0	0.13
MMC	0.00005	48	200	-	-	20*	17*	0.25

¹DMSO

²800 cells were analysed in each group

³Cell confluency, representing cytotoxicity, was measured with Monocellater^TM

⁴Number of metaphase per 500 cells was scored in each dish in order to select the highest dose enable to analyze chromosomes

*Significantly different from solvent control at p<0.01 by Fisher’s exact test

**Purity was 98.2% wt%. Diacetin (0.9%) was contained as impurity

MMC = Mitomycin C, positive control group

 

Table 2. Chromosome analysis of Chinese hamster lung cells (CHL/IU) treated with glycerol triacetate (TA)** with and without S9 mix. 

Group	S9 Mix	Concen- tration (mg/mL)	Expo-sure (h)	No. of cells ana-lyzed	Cyto-toxicity^3 (%)	Mito- tic index^4 (%)	Aberrant cells in %		Poly-ploid^2 (%)
							incl. gaps	excl. gaps
Non-treatment				200	-	-	0.0	0.0	0.0
Solvent^1	-	0	6-(18)	200	100	-	0.0	0.0	0.13
Triacetin	-	0.55	6-(18)	200	100.5	-	0.5	0.5	0.63
Triacetin	-	1.1	6-(18)	200	99.5	-	1.0	1.0	0.0
Triacetin	-	2.2	6-(18)	200	90.5	9.2	0.0	0.0	0.25
CPA	-	0.005	6-(18)	200	-	-	0.5	0.5	0.50
Solvent^1	+	0	6-(18)	200	100	-	0.0	0.0	0.25
Triacetin	+	0.55	6-(18)	200	109	-	0.0	0.0	0.5
Triacetin	+	1.1	6-(18)	200	104.5	-	0.5	0.5	0.25
Triacetin	+	2.2***	6-(18)	200	25.5	1.6	43.5*	42.0*	0.26
CPA	+	0.005	6-(18)	200	-	-	45.5*	45*	0.13

¹DMSO

²800 cells were analysed in each group

³Cell confluency, representing cytotoxicity, was measured with Monocellater^TM

⁴Number of metaphase per 500 cells was scored in each dish in order to select the highest dose enable to analyze chromosomes

*Significantly different from solvent control at p<0.01 by Fisher’s exact test

**Purity was 98.2 % wt%. Diacetin (0.9%) was contained as impurity

***Medium colour was changed to yellow, which showed lowering of pH, after treatment

CPA = Cyclophosphamide, positive control group

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

.