Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

From September 15, 2016 to October 19, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity/composition: 10.0% dry matter
Appearance: whitish liquid

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate (based on range finding dose study and cytotoxicity).
The highest concentration of the test substance used in the mutation assays was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Vehicle / solvent:

Ethanol for the test substance (and DMSO or saline for positive controls)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol, DMSO or saline

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191(without metabolic activation), 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

- Following dose range findings studies, the test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix.
- Exposure: 48 ± 4h (+ a pre-incubation of 30 min if needed)

Rationale for test conditions:

- Based on the most recent OECD and EC guidelines
- Dose range finding studies
- First mutation experiments

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test substance was considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control. b) The negative response should be reproducible in at least one follow up experiment.
- A test substance was considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains except in S. typhimurium TA1537 in the absence of S-9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA1535, TA1535, TA98 and TA100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains except in E. coli WP2uvrA

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA1535, TA1535 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay.
- In the first mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain TA1537 in the absence of S9-mix.
- In the second mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Precipitation was observed in the absence and presence of S9-mix. Due to the composition of the test substance, no revertant colonies or bacterial background lawn could be determined at the highest dose level tested. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA. Since in the second mutation test, due to toxicity, only two or three analysable dose levels were present in the tester strains TA1537 (absence of S9-mix), and TA1535 and TA98 (absence and presence of S9-mix), an additional experiment was performed.
- In this third mutation experiment, the test substance was tested at concentration ranges of 0.54 to 164 µg/plate in the absence of S9-mix and at a range of 1.7 to 512 µg/plate in the presence of S9-mix. The test substance was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Based on the results of this study it is concluded that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Remarks on result:

other: first experiment

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate for 48 ± 4 h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. In the first mutation experiment, the test substance was tested up to concentrations of  5000 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates. Cytotoxicity was observed in all tester strains in the absence and presence of S9 -mix, except in tester strain TA1537 in the absence of S9 -mix. In the second mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Precipitation was observed in the absence and presence of S9 -mix. Due to the composition of the test substance, no revertant colonies or bacterial background lawn could be determined at the highest dose level tested. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA. Since in the second mutation test, due to toxicity, only two or three analysable dose levels were available for the tester strains TA1537 (absence of S9-mix), and TA1535 and TA98 (absence and presence of S9-mix), an additional experiment was performed. In this third mutation experiment, the test substance was tested at concentration ranges of 0.54 to 164 µg/plate in the absence of S9-mix and at a range of 1.7 to 512 µg/plate in the presence of S9-mix. Cytotoxicity was observed in all three tester strains. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2016).  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 19, 2016 to November 11, 2016

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Specific details on test material used for the study:

Batch no.: RE 10-7
Purity: 92.75%
Appearance: liquid yellow paste
Solubility: the test substance was dissolved in ethanol

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Cultured peripheral human lymphocytes
- Whole blood samples obtained from healthy subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

First assay: 0 - 270 and 0 - 260 µg/mL without and with metabolic activation, respectively
Second assay: 0 - 200 and 0 - 150 µg/mL without metabolic activation for a 24 and 48 h exposure time, respectively
(The highest tested concentrations were selected based on dose range finding tests)

Vehicle / solvent:

Ethanol and Hanks’ Balanced Salt Solution

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

ethanol and HBSS

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- In the first cytogenetic assay, the test substance was tested up to 270 and 260 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9fraction. Appropriate toxicity was reached at these dose levels.
- In the second cytogenetic assay, the test substance was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

Rationale for test conditions:

- Guidelines
- Dose-range finding study
- Cells and solubility of the test substance

Evaluation criteria:

- Number of chromosome aberrations
(Chromosome aberrations are generally evaluated in the first post-exposure mitosis (24 h after exposure). However, since the appearance of the first post-exposure mitosis could be considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure to cover the interval in which maximum aberration frequency was expected.)
- Criteria:
A test substance is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.  
b) The increase is dose related when evaluated with a trend test.  
c) Any of the results are outside the 95% control limits of the historical control data range.
A test substance is considered negative (not clastogenic) in the chromosome aberration test if:  
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range. In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.  

Statistics:

- Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data
- Fisher’s exact test, onesided, p < 0.05

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

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:

The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. It is concluded that this test is valid.

Conclusions:

Under the study conditions, the substance was not clastogenic in the chromosomal aberration assay in cultured peripheral human lymphocytes.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the substance, according to OECD Guideline 473, in compliance with GLP. Two experiments were performed on cultured peripheral human lymphocytes. In the first cytogenetic assay, the test substance was tested up to 270 and 260 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9 fraction. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test substance was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 µg/mL for a 48 h continuous exposure time and a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The test substance did not induce any statistically significant or biologically relevant increase in the number of chromosome aberrations in the absence and presence of S9-mix, in either of the two experiments. No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of  S9 -mix. The number of cells with chromosome aberrations found in the solvent control cultures was within 95% limits of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within 95% limits of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the substance was not clastogenic in the chromosomal aberration assay in cultured peripheral human lymphocytes (Verbaan, 2017).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 1824 2016 to January 02, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

in the study plan: the survival in the highest dose tested. The study integrity was not adversely affected by the deviation

GLP compliance:

yes

Type of assay:

other: in vitro gene mutation study in mammalian cells

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity: 10.0% (a correction factor of 10 was therefore used)
Appearance: whitish liquid

Target gene:

Thymidine-kinase locus (TK-locus)
(The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect. Cells deficient in thymidine kinase (TK), due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT)).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

cell density was kept below 1 x 106 cells/mL

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:

0 - 110 µg/L (based on a dose range finding test, the solubility of the test substance and the cytotoxicity observed)

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

The test was performed in the absence of S9-mix with 3 (with 10E06 cells/mL) and 24-hour (with 1.25E05 cells/mL) treatment periods and in the presence of S9-mix with a 3 h treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).
In the first experiment, effect of the test substance was evaluated up to the dose levels of 80 and 90 µg/mL in the absence and presence of S9-mix, respectively giving Relative Total Growth (RSG) values of 10 and 43%. Above this dose level the RSG was below the acceptable limit of 10% for both absence and presence of S9-mix. The incubation time was 3 h. No precipitation was observed up to the concentration of 90 µg/mL.
In the second experiment, effect of the test substance was evaluated up to the dose level of 110 μg/mL giving a RTG of 63%. Above this dose level the RSG was below the acceptable limit of 10%. The incubation time was 24 hours. No precipitation was observed up to the concentration of 110 µg/mL.
For expression of the mutant phenotype, cells were cultured for 2 days after the treatment period. During this culture period at least 4.0E06 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CE day 2) and the mutation frequency (MF).

Rationale for test conditions:

Dose range finding test
Solubility of the test substance

Evaluation criteria:

- Determination of the mutant colonies, calculation of the survival or viability, calculation of the mutation frequency (MF) and cloning efficiency (CEday2)
- 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% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300E10-6). At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control should have an increase in the small colony MF of at least 150E10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150E10-6).

Statistics:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT 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.
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: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
It is concluded that the test substance was not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Conclusions:

Under the study conditions, the substance was not mutagenic in mouse lymphoma L5178Y cells.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the substance according to OECD Guideline 490, in compliance with GLP. Two experiments were performed on mouse lymphoma L5178Y cells at the thymidine-kinase locus (TK-locus). The test was performed in the absence of S9 -mix with 3 (with 10E+06 cells/mL) and 24 h (with 1.25E+05 cells/mL) treatment periods and in the presence of S9 -mix with a 3 h treatment period. In the first experiment, effect of the test substance was evaluated up to the dose levels of 80 and 90 µg/mL in the absence and presence of S9-mix, respectively giving Relative Total Growth (RSG) values of 10 and 43%. Above this dose level the RSG was below the acceptable limit of 10%. The incubation time was 3 h. No precipitation was observed up to the concentration of 90 µg/mL. In the second experiment, effect of the test substance was evaluated up to the dose level of 110 μg/mL giving a RTG of 63%. Above this dose level the RSG was below the acceptable limit of 10%. The incubation time was 24 h. No precipitation was observed up to the concentration of 110 µg/mL. For expression of the mutant phenotype, cells were cultured for 2 d after the treatment period. During this culture period at least 4.0E06 cells were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). In the absence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment and in the presence of S9 -mix. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the substance was not mutagenic in the mouse lymphoma L5178Y cells (Verspeek-Rip, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the in vitro genetic toxicity of the substance according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate for 48 ± 4 h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. In the first mutation experiment, the test substance was tested up to concentrations of  5000 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates. Cytotoxicity was observed in all tester strains in the absence and presence of S9 -mix, except in tester strain TA1537 in the absence of S9 -mix. In the second mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Precipitation was observed in the absence and presence of S9 -mix. Due to the composition of the test substance, no revertant colonies or bacterial background lawn could be determined at the highest dose level tested. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA. Since in the second mutation test, due to toxicity, only two or three analysable dose levels were available for the tester strains TA1537 (absence of S9-mix), and TA1535 and TA98 (absence and presence of S9-mix), an additional experiment was performed. In this third mutation experiment, the test substance was tested at concentration ranges of 0.54 to 164 µg/plate in the absence of S9-mix and at a range of 1.7 to 512 µg/plate in the presence of S9-mix. Cytotoxicity was observed in all three tester strains. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2016).  

A study was conducted to determine the in vitro genetic toxicity of the substance, according to OECD Guideline 473, in compliance with GLP. Two experiments were performed on cultured peripheral human lymphocytes. In the first cytogenetic assay, the test substance was tested up to 270 and 260 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9 fraction. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test substance was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 150 µg/mL for a 48 h continuous exposure time and a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The test substance did not induce any statistically significant or biologically relevant increase in the number of chromosome aberrations in the absence and presence of S9-mix, in either of the two experiments. No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of  S9 -mix. The number of cells with chromosome aberrations found in the solvent control cultures was within 95% limits of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within 95% limits of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the substance was not clastogenic in the chromosomal aberration assay in cultured peripheral human lymphocytes (Verbaan, 2017).

A study was conducted to determine the in vitro genetic toxicity of the substance according to OECD Guideline 490, in compliance with GLP. Two experiments were performed on mouse lymphoma L5178Y cells at the thymidine-kinase locus (TK-locus). The test was performed in the absence of S9 -mix with 3 (with 10E+06 cells/mL) and 24 h (with 1.25E+05 cells/mL) treatment periods and in the presence of S9 -mix with a 3 h treatment period. In the first experiment, effect of the test substance was evaluated up to the dose levels of 80 and 90 µg/mL in the absence and presence of S9-mix, respectively giving Relative Total Growth (RSG) values of 10 and 43%. Above this dose level the RSG was below the acceptable limit of 10%. The incubation time was 3 h. No precipitation was observed up to the concentration of 90 µg/mL. In the second experiment, effect of the test substance was evaluated up to the dose level of 110 μg/mL giving a RTG of 63%. Above this dose level the RSG was below the acceptable limit of 10%. The incubation time was 24 h. No precipitation was observed up to the concentration of 110 µg/mL. For expression of the mutant phenotype, cells were cultured for 2 d after the treatment period. During this culture period at least 4.0E06 cells were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance, the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF). In the absence of S9 -mix, the test substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment and in the presence of S9 -mix. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the substance was not mutagenic in the mouse lymphoma L5178Y cells (Verspeek-Rip, 2017).

Justification for classification or non-classification

Based on the in vitro genetic toxicity studies with the substance, no classification for genetic toxicity is warranted according to EU CLP (EC 1272/2008) criteria.