Phosphoric acid, mono- and di-C6-12-(even numbered)-alkyl esters, potassium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 06, 2017 to December 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: White solid
Batch: RE 14-6
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 July 2018 (expiry date)

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: See remarks

Remarks:

rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: See remarks

Remarks:

rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galacto se metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Metabolic activation:

with and without

Metabolic activation system:

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 susbtance 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
DMSO or saline for positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or saline

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (wthout metabolic activation) and 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 experiment

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 TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not 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, a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strain TA100 at dose levels of 512 and upwards and 1600 μg/plate in the absence and presence of S9-mix, respectively. 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 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this 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 tester strains in the absence and presence of S9-mix.

- In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to concentrations of 5000 µg/plate in the tester strain WP2uvrA, in the pre-incubation assay. The test substance did not precipitate on the plates at these dose levels. 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.

- Due to severe cytotoxicity an additional mutation experiment was performed in the absence of S9-mix. In the additional experiment the test substance was tested at a concentration range of 0.06 to 17 µg/plate in tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 0.55 to 164 µg/plate in tester strain WP2uvrA. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all 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. These results were confirmed in a follow-up experiment.

- Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Conclusions:

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

Executive summary:

A study was conducted to determine the mutagenic potential 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 preincubation 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, and also exposed to the negative or positive control substances for 48 ± 4 h (plus a pre-incubation time of 30 min if needed). In the dose range finding study, the test substance was initially tested up to 5000 μg/plate in the tester strains TA100 and WP2uvrA through a direct plate assay. Cytotoxicity was evidenced in TA100 in the absence and presence of S9 -mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to concentrations of 5000 µg/plate in the tester strain WP2uvrA, in the pre-incubation assay. The test substance did not precipitate on the plates at these dose levels. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Due to severe cytotoxicity an additional mutation experiment was performed in the absence of S9-mix. In the additional experiment the test substance was tested at a concentration range of 0.06 to 17 µg/plate in tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 0.55 to 164 µg/plate in tester strain WP2uvrA. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. 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. These results were confirmed in an independently repeated experiment. 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. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verbaan, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

A study was conducted to determine the mutagenic potential 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 preincubation 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, and also exposed to the negative or positive control substances for 48 ± 4 h (plus a pre-incubation time of 30 min if needed). In the dose range finding study, the test substance was initially tested up to 5000 μg/plate in the tester strains TA100 and WP2uvrA through a direct plate assay. Cytotoxicity was evidenced in TA100 in the absence and presence of S9 -mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to concentrations of 5000 µg/plate in the tester strain WP2uvrA, in the pre-incubation assay. The test substance did not precipitate on the plates at these dose levels. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Due to severe cytotoxicity an additional mutation experiment was performed in the absence of S9-mix. In the additional experiment the test substance was tested at a concentration range of 0.06 to 17 µg/plate in tester strains TA1535, TA1537, TA98 and TA100 and at a concentration range of 0.55 to 164 µg/plate in tester strain WP2uvrA. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. 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. These results were confirmed in an independently repeated experiment. 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. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verbaan, 2017).

Justification for classification or non-classification

Based on the in vitro genetic toxicity study with the test substance, no conclusion on classification for genetic toxicity could be derived according to EU CLP (1272/2008) criteria.