Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Genetic toxicity: in vitro

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:

13-Mar-2012 to 15-May-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

EC Number:

615-240-7

Cas Number:

710292-85-6

Molecular formula:

Not available for this UVCB

IUPAC Name:

Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Test material form:

other: waxy solid

Details on test material:

- Name of test material (as cited in study report): HK 128
- Substance type: Red-violet waxy solid
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 1, 10, 30, 50, 80, 90, 100 and 120 µg/mL
With S9-mix, 3 hours treatment: 1, 10, 30, 100, 150, 200, 250 and 350 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 3, 10, 30, 50, 70, 80, 90 and 100 µg/mL
With S9-mix, 3 hours treatment: 1, 3, 10, 100, 200, 300, 350 and 400 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide 10 µg/mL

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 70 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and above in the absence of S9-mix at the 3 hour and prolonged treatment period. Precipitation in the exposure medium was observed at the dose level of 333 µg/mL in the presence of S9-mix..

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment, HK 128 was tested up to cytotoxic levels of 85 and 97% in the absence and presence of S9-mix, respectively.

In the second experiment, HK 128 was tested up to cytotoxic levels of 87 and 73% in the absence and presence of S9-mix, respectively.

Applicant's summary and conclusion

Conclusions:

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. HK 128 is not mutagenic in the mouse lymphoma L5178Y test system

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 7.5- and 15 -fold for in the absence of S9-mix, and by 15- and 14-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, HK 128 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, HK 128 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.