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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 13, 1978 to January 24, 1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted similar or equivalent to OECD guideline 476
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trimethylolpropane triacrylate
- Physical state: Clear colourless liquid

Method

Target gene:
Thymidine Kinase (TK)

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: L5178Y mouse lymphoma cells TK (+/-) derived from the Fischer L5178Y line of Dr. Donald Clive.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary toxicity test:
0.004875 to 5 nL/mL (without S9); 0.004875 to 40 nL/mL (with S9).

Mutation test:
Test 1: 0.078 to 1.25 nL/mL without S9; 1.150 to 2.50 nL/mL with S9
Test 2: 0.150 to 1.00 nL/mL without S9; 1.250 to 10.00 nL/mL with S9
Test 3: 1.00 to 2.5 nL/mL without S9; 2.00 to 20 nL/mL with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
absence of S9 mix

Migrated to IUCLID6: 0.5 µL/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
presence of S9 mix

Migrated to IUCLID6: 0.3 µL/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In suspension

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48-72 h
- Selection time (if incubation with a selection agent): 10 d

NUMBER OF REPLICATIONS: Duplicate

Evaluation criteria:
A compound is considered mutagenic in this assay if:
- A dose-response relationship is observed in 3 of the 5 dose levels employed.
- The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
- The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.
Statistics:
No data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity testing without activation indicated variable toxicity and complete lethality at 1.25 to 2.5 µL/mL. Three trials of the mutation assay are reported in which the inclusive applied dose ranges were 0.004875 to 5 µL/mL without activation, and 0.004875 to 40 µL/mL with activation. Dose levels showing excessive or insufficient toxicity to cell growth were eliminated from further testing
in order to select five doses for completion of the assay that would fall within the range of cytotoxicities where any mutagenic activity is normally observed.

MAIN STUDY:
Without metabolic activation - Significant increase in mutant frequency (3.6 times the background) was observed only at highest tested dose of 1.25 nL/mL in the first trial. This dose also showed high toxicity (19.1% relative growth). In the second trial, the same pattern of response was obtained. Significant increase in mutant frequency (4.2 times the background level) was observed only at the highest tested dose of 1.0 nL/mL (with 32.7% relative growth). Because the background frequency was unusually low in this trial, the assay was repeated again. In the third trial higher doses were tested and more extreme toxicities were obtained. Mutagenic activity was observed at the two highest doses (1.0 and 2.5 nL/mL) and not at lower doses.
 
With metabolic activation - The results of first trial were inconclusive. In the second trial, no increase in mutant frequency was found for the 1.25 to 10.0 nL/mL dose range, in spite of the high toxicity at 10.0 nL/mL (14.9% relative growth). Because the background and positive control mutant frequency was unusually low in this trial, the assay was repeated again. The repeat assay showed a 6.4-fold increase in mutant frequency at 20 nL/mL. This dose was highly toxic (4.8% relative growth), whereas little toxicity was obtained with the other tested dose levels.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.
Executive summary:

A study was conducted to assess the mutagenic potential of trimethylolpropane triacrylate on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was equivalent or similar to OECD guideline 476.

Three independent tests in the absence of exogenous metabolic activation and three independent tests in the presence of S9 mix were carried out using the concentrations based on results of the preliminary cytotoxicity trials.

 

In the absence of S-9 mix, significant increase in mutant frequency (3.6 times the background) was observed only at highest tested dose of 1.25 nL/mL in the first trial. This dose also showed high toxicity (19.1% relative growth). In the second trial, the same pattern of response was obtained. Significant increase in mutant frequency (4.2 times the background level) was observed only at the highest tested dose of 1.0 nL/mL (with 32.7% relative growth). Because the background frequency was unusually low in this trial, the assay was repeated again. In the third trial higher doses were tested and more extreme toxicities were obtained. Mutagenic activity was observed at the two highest doses (1.0 and 2.5 nL/mL) and not at lower doses.

 

In the presence of S-9 activation, the results of first trial were inconclusive. In the second trial, no increase in mutant frequency was found for the 1.25 to 10.0 nL/mL dose range, in spite of the high toxicity at 10.0 nL/mL (14.9% relative growth). Because the background and positive control mutant frequency was unusually low in this trial, the assay was repeated again. The repeat assay showed a 6.4-fold increase in mutant frequency at 20 nL/mL. This dose was highly toxic (4.8% relative growth), whereas little toxicity was obtained with the other tested dose levels.

 

In conclusion, trimethylolpropane triacrylate induced an increase in mutations at the TK locus in L5178Y mouse lymphoma cells in the dose range of 1.0 to 2.5 nL/mL without activation and at 20.0 nL/mL with microsomal activation. This mutagenic response was associated only with doses that were moderately to highly toxic. Trimethylolpropane triacrylate was considered to be mutagenic to L5178Y cells with and without metabolic activation.