Propanoic acid, 2-[4-[4,6-bis([1,1'-biphenyl]-4-yl)-1,3,5-triazin-2-yl]-3-hydroxyphenoxy]-, isooctyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material itself did not show mutagenic or clastogenic activity in vitro in a bacterial reverse mutagenicity assay and a chromosome aberration test (according to OECD guidelines 471 and 473, GLP). Two analgue substances did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (according OECD guideline 476, GLP). Therefore, the test substance is not considered to be genotoxic. 

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

Result #1refers to CAS 204583-39-1; Result #2 refers to CAS 446824-06-2

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-Nov-2002 to 30-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 473)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted on 21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

19 March 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human, cultured in vitro

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction from Aroclor 1254 induced rat liver (S9 mix)

Test concentrations with justification for top dose:

First test: 15.6, 31.3 and 62.5 µg/mL (with and without S9 mix)
Second test:
62.5, 125 and 500 µg/mL ( without S9 mix)
250, 500 and 1000 µg/mL ( with S9 mix)
The following concentration were selected for metaphase analysis: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL (first and second test with and without S9 mix)

Vehicle / solvent:

- Solvent used: acetone
- Justification for choice of solvent:
Prior to commencing testing, the solubility of the test substance in solvents compatible with the test system was assessed. The test item was found to be soluble in Acetone at 465 mg/mL. On dosing at 1% (v/v) into aqueous tissue culture medium, precipitates were observed at final concentrations of 290.63 µg/mL and above. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 µg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 1000 µg/mL, in order to include at least two precipitating dose levels in the concentration range.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL (3-hour treatment) 0.1 µg/mL (Continuous treatment)

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Positive controls:

yes

Remarks:

10 µg/mL

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® (Sigma) to each cultare at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tabe and centrifuged for 5 minutes at 500 g. The cell pellets
were treated with a hypotonic solution (0.075M KCl prewarmed at 37 °C). After a 10 minute period of hypotonic incubation at 37 °C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid)
Second test:
- Exposure duration: continuously (without S9 mix), 3 hours (with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of chromosome aberrations, polyploidy and endoreplication:
Prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum concentration was used as the highestdose level for the metaphase analysis. The intermediate and low dose levels were also selected. One hundred metaphase figures were examined from each culture. This number was reduced in cultures showing a high level of aberrant cells. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44-48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.
The incidence for polyploidy metaphase cells, out of 500 metaphase cells, was determined quantitatively for all cultures used in the analysis for chromosomal aberrations. The number of aberrant and polyploid metaphase cells in each treatment group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher, 1973).

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (P < 0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration. The increases exceed the negative control range, taken at the 99% confidence limit. The increases are reproducible between replicate cultures. The increases are not associated with large changes in osmolality of the treatment medium or
extreme toxicity. Evidence of a dose-relationship is considered to support the conclusion. A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level. A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

An assay is considered to be acceptable if the negative and positive confrol values lie within the current historical control range.

Species / strain:

lymphocytes: human, cultured in vitro

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: low
- Precipitation: at 1000 µg/mL (without S9 mix)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 64% of the solvent control value at 1000 µg/mL. In the presence of S9 mix, the test item failed to cause a significant reduction in the mitotic index compared to the solvent control value. Dose levels were selected accordingly.

Conclusions:

The test substance showed no evidence of clastogenic activity in a in vitro cytogenetic test system.

Executive summary:

A GLP-compliant study according to OECD TG 473 was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived firom rat livers. Two hours before the end of the incubation period, cell division was arrested using Colcemid , the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the first test (with and without S9 mix, 3 hours treatment) and in the second test (without S9 mix, 20 hours continuous treatment or in presence of S9 mix, 3 hours treatment) the test material caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any dose level, when compared with the solvent control, in either test. A quantitative analysis for polyploidy was made in cultures treated with the negative control and highest dose level. No statistically significant increases in the proportion of polyploid cells were seen.

It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-Nov 2002 to 21-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study (according OECD 471)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 12th July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8747, 24 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal fraction S9 mix from Aroclor 1254 induced liver of male Sprague-Dawley derived rats

Test concentrations with justification for top dose:

5; 15; 50; 150; 500; 1500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent:
The solubility of the test item in water was low (-100 µg/L). Its solubility was, therefore, assessed at 50 mg/mL in dimethyl sulphoxide (DMSO), in which it dissolved following sonication for 45 minutes.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strains TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

50 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

1 µg/plate for strain TA98

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

0.05 µg/plate for strain WP2uvrA/pKM101

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-fiiryl) acrylamide

Remarks:

Without metabolic activation

Positive controls:

yes

Remarks:

2 µg/plate for strain TA1535; 10 µg/plate for strain WP2MuvrA/pKM101

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Positive controls:

yes

Remarks:

5 µg/plate for strains TA 153 7, TA98 and TA 100

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Range-finding test: standard plate incorporation assay (first test)
The test substance was added to cultures of the five tester strains at seven concentrations separated by ca. half-log10 intervals. The highest concentration tested was 50 mg/mL in the chosen solvent, which provided a final concentration of 5000 µg/plate. The vehicle control was the chosen solvent, DMSO. The appropriate positive controls were also included. Aliquots of 0.1 mL of the test dilution, positive control or negative control were placed in glass vessels. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10 hour bacterial culture and 2 mL of agar containing histidine (0.5 mM), biotin (0.5 mM) and tryptophan (0.5 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Three Petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer. All plates were incubated at 37 °C for ca. 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.
- Second test:
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay. The top concentration chosen was again 5000 µg/plate, but only 5 concentrations were used.

DURATION
- Preincubation period: 30 minutes
- Exposure duration:
First test (range-finding): 72 hours
Second test: 30 minutes

NUMBER OF REPLICATIONS: three per concentration in each of two independent experiments

DETERMINATION OF CYTOTOXICITY
Any toxic effects would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

If exposure to the test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship, it will be considered to exhibit mutagenic activity in this test system. If exposure to the test substance does not produce a reproducible increase in revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system.If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Statistics:

The statistical procedures used will be those described by Mahon et al (1989) and will usually be Dunnett's test followed, if appropriate, by trend analysis. The mean number and standard deviation of revertant colonies were calculated for all groups. The means for all treatment groups were compared with those obtained for the solvent/vehicle control groups

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration in either the presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test item. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

Conclusions:

The test substance showed no evidence of mutagenic activity in a bacterial system.

Executive summary:

The mutagenic potential of the test substance was assessed in a GLP-compliant in vitro study according to OECD TG 471. Histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TAI535, TAI537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA, were exposed to the test item diluted in dimethyl sulphoxide (DMSO). Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay; the second involved a pre-incubation stage. Concentrations up to 5000 µg/plate were tested. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration in either mutation test.

It is concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Procedure and observations

In the in vitro assessment of the mutagenic potential of the test substance, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TAI535, TAI537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA, were exposed to the test item diluted in dimethyl sulphoxide (DMSO). Two independent mutation tests were performed in the presence and absence of liver preparations firom Aroclor 1254-treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay; the second involved a pre-incubation stage. Concentrations up to 5000 µg/plate were tested. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration in either mutation test.

 

The second study was performed to assess the ability of the test material to induce chromosomal aberrations in human lymphocytes cultured in vitro. Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the presence and absence of S9 mix derived firom rat livers. Two hours before the end of the incubation period, cell division was arrested using Colcemid , the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. In the first test (with and without S9 mix, 3 hours treatment) and in the second test (without S9 mix, 20 hours continuous treatment or in presence of S9 mix, 3 hours treatment) the test material caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations.

 

The test substance was not examined for its potential to induce gene mutations in mammalian cells. Reliable experimental data on two read across substance are available (see justification attached to IUCLID section 7.6.1 and 13):

In a GLP conform study according to OECD guideline 476 the potential of the structural analogue (CAS 204583-39-1) to induce mutations V 79 cells was assessed. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Due to limited solubility of the test item in a suitable solvent the highest concentration of the test item in the pre-experiment was 2666.6 mg/L resulting in strong test item induced precipitation at the end of the treatment period. As no cytotoxicity was observed the highest concentration applied in the main experiments was far above the limit of solubility of the test item in culture medium. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

The potential of the structural analogue (CAS 446824-06-2) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was determined in an in vitro assay according to OECD TG 476 and GLP. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (3600 µg/mL) was limited by the solubility properties of the test item. The test item was dissolved in acetone. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.

 

Conclusion

The test substance or analogue substance respectively, showed no evidence of mutagenic activity in bacterial or mammalian cells and it has shown no evidence of clastogenic activity in vitro. The substance is therefore not considered to be genotoxic.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.