Triacetin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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: GLP - Guideline study with acceptable restrictions. Lack of test material details and no historical control data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

lack of test material details and no historical control data.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

905-964-4

EC Number:

905-964-4

IUPAC Name:

905-964-4

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: colourless liquid
- Chemical denomination: glycerol monoacetate
- Analytical purity: assumed to be 100%
- Lot/batch No.: 38988
- Expiration date of the lot/batch: 1996-07-30
- Storage condition of test material: room temperature

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 buffered with sodium bicarbonate containing sodium pyruvate/Synperonic F68, L-glutamine and gentamicin

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Preliminary toxicity test
50, 150, 300, 625, 1250, 2500, 3750 and 5000 µg/mL
Experiment I/II
Without S9 mix: 300, 625, 1250, 2500, 3750 and 5000 µg/mL
With S9 mix: 700, 625, 1250, 2500, 3750 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the miscibility of the test material in sterile deionized water was asses prior to the testing. It was confirmed that the test material was fully miscible with water at a concentration of 500 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate, 10 µg/mL in water, without S9; 20-methylcholanthrene, 2.5 µg/mL in DMSO, with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time: 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-14 days

SELECTION AGENT: Selective medium consisted of cloning medium containing 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The criteria for a positive response were:
An increase in mutant frequency in treated cultures of at least 100 relative to the concurrent control. The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance. Evidence of a dose relationship over at least two dose levels, in any increase in mutant frequency. Demonstration of reproducibility in any increase in mutant frequency. The observed increases in mutant frequency must lie outside the historical control range with a corresponding RTG of 20%.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al (1989).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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
- Effects of pH: a decrease in pH from the control level of pH 7.5 to pH 6.0 at 5000 µg/mL was observed in both tests.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, treatment with 50-5000 µg/mL in the absence and presence of S9 mix resulted in relative growth in suspension of 129-107% and 104-85% respectively compared to the solvent control.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment I - in the absence of S9-mix.

Concentration	Relative Total Growth [%]	Growth in Suspension [%]	Mutants per 1E+06 surviving cells
[µg/mL]
0 (DMSO)	100	100	137
1250	86	86	128
2500	88	88	140
3750	88	86	118
5000	90	99	148
MMS, 10	46	84	657***

MMS = Methyl methane sulphonate

*** Significantly different from control P < 0.001

Table 2: Experiment II - in the absence of S9-mix. 

Concentration	Relative Total Growth [%]	Growth in Suspension [%]	Mutants per 1E+06 surviving cells
[µg/mL]
0 (DMSO)	100	100	174
1250	86	97	187
2500	104	99	179
3750	76	85	189
5000	92	97	176
MMS, 10	63	87	469***

MMS = Methyl methane sulphonate

*** Significantly different from control P < 0.001

Table 3: Experiment I - in the presence of S9-mix. 

Concentration	Relative Total Growth [%]	Growth in Suspension [%]	Mutants per 1E+06 surviving cells
[µg/mL]
0 (DMSO)	100	100	172
1250	100	97	133
2500	91	90	144
3750	83	78	169
5000	85	78	167
20-MC, 2.5	19	37	881***

20 -MC: 20 -Methylcholanthrene

*** Significantly different from control P < 0.001

Table 4: Experiment II - in the absence of S9-mix.

Concentration	Relative Total Growth [%]	Growth in Suspension [%]	Mutants per 1E+06 surviving cells
[µg/mL]
0 (DMSO)	100	100	128
1250	89	93	120
2500	79	82	130
3750	71	78	125
5000	62	79	131
20-MC, 2.5	16	34	775***

20 -MC: 20 -Methylcholanthrene

*** Significantly different from control P < 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative