5-butoxy-2-[4-(4-butoxy-2-hydroxyphenyl)-6-(2,4-dibutoxyphenyl)-1,3,5-triazin-2-yl]phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test in E. coli and Salmonella (OECD 471) and a chromosome aberration assay (OECD 473) in mammalian cells was performed to evaluate the genotoxic potential of the test item. The substance did neither induce point mutations in bacterial DNA nor chromosomal damage in mammalian cells.

Moreover, an HPRT (OECD 476) conducted on a structural analogue is available. The read across substance and its metabolites did not show any mutagenic activity in this forward mutation system conducted in chinese hamster lung fibroblasts.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His: Salmonella
Trp: E. coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-Naphthoflavone-induced liver S9 mix from rats

Test concentrations with justification for top dose:

incorporation test (experiment I) and the preincubation test (experiment II) (with and without metabolic activation): 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA 1535, TA 100: sodium azide, NaN3, TA1537, TA98: 4-nitro-o-phenylene-diamine, 4-NOPD, WP2 uvrA: methyl methane sulfonate, MMS

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 60min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: all colonies counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results:
A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

other: TA 1535, TA 1537, TA 98, and TA 100, WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at higher concentrations

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 - 5000 ug/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 ug/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, for negative control without metabolic activation, solvent control wihtout metabolic activation, positive control without metabolic activation

Conclusions:

The reverse bacterial mutation test did not reveal a genotoxicity potential.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Without S-9 mix:
Experiment I: 3.2 - 1000 µg/ml
Experiment II: 1.0 - 1000 µg/ml

With S-9 mix:
Experiment I: 3.2 - 1000 µg/ml
Experiment II: 1.0 - 1000 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Without S-9 mix:
Experiment I: 3.2 - 1000 µg/ml; Exposure period = 4 hours ; Recovery = 14 hours; preparation interval = 18 hours
Experiment II: 1.0 - 1000 µg/ml; Exposure period =18; Preparation interval= 28 hours
10.0 - 1000 µg/ml; Exposure period = 28 hours; Preparation interval= 28 hours

With S-9 mix:
Experiment I: 3.2 - 1000 µg/ml; Exposure period = 4 hours ; Recovery = 14 hours; preparation interval = 18 hours
Experiment II: 1.0 - 1000 µg/ml; Exposure period = 4 hours; Recovery = 24 hours; Preparation interval= 28 hours

16 h and 26 h, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa.
Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

Evaluation criteria:

The chromosome aberration assay performed is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data.

A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed. A test item is classified as non-mutagenic if
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (p < 0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation: Precipitation of the test item in culture medium was observed at all applied concentrations.
However, 4 h after start of treatment, precipitation was observed at 31.6 µg/ml and above. Although no clear toxicity was observed, 1000 µg/ml were chosen as top concentration in experiment I for the evaluation of cytogenetic damage, to confirm the toxicity observed in the range-finding experiment. In experiment Il just as in experiment I no clear toxicity was observed. Therefore, the highest evaluated concentration was chosen considering the observed precipitation at 31.6 µg/ml and above as recommended in the OECD Guideline 473.

RANGE-FINDING/SCREENING STUDIES:
A pre-test on cell growth inhibition with 4 h and 24 h treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - 3.33. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, with control data V79 cell line since 1993 (positive and negtive control)

Conclusions:

The in vitro chromosome aberration test in V79 cells did not reveal a genotoxicity potential at all concentrations. No cytotoxicity was observed even at concentrations that did produce precipitation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

mammalian cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the results of two independently performed experiments in the analogue substance and under the given experimental conditions, it is concluded that the test item and its metabolites did not show any mutagenic activity in this forward mutation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are two reliable in vitro studies available to assess the potential of the test substance for gene mutations in bacteria and cytogenicity in mammalian cells.

In a GLP conform study according to OECD guideline 471, the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA was investigated. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. In experiment II, toxic effects, evident as a reduction in the number of revertants, were observed at the higher concentrations in strains TA 1537, TA 98, TA 100, and WP2 uvrA in the presence of metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In a GLP conform study according to OECD guideline 473, the test substance, formulated in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation with respect to the OECD Guideline 473.No clear toxic effects, either indicated by reduced mitotic indices or indicated by reduced cell numbers were observed in the absence and the presence of S9 mix. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

There are no data on the mutagenic activity (HPRT test) of the test substance available. However, another structural analogue source substance was tested for this endpoint and the result can be transferred to the test substance in order to fill this data gap.

In detail, a GLP-compliant study according to OECD 476 was performed with another analogue substance. Based on preliminary toxicity test four concentrations were selected for the original mutagenicity experiment ranging from 18.52 to 500 µg/ml in the presence and absence of metabolic activation. 500 µg/ml was the highest attainable concentration due to the solubility limit inthe vehicle.The mutagenic activity was measured in the presence and absence of metabolic activation. Although the original experiment revealed statistically significant differences at some of the concentrations, both in the absence and in the presence of metabolic activation, the criteria for a positive response were not fullfilled. The increase was not concentration dependent in both parts and the number of mutant colonies differed from the respective number of the negative control by much less than the required 20. Furthermore, the effects were not reproducible. The positive controls induced a clear increase in mutant frequency. Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.