Potassium hexadecyl hydrogen phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two reverse mutation bacterial test (Ames tests) the test substance was proved to be not mutagenic. Gene mutation toxicity study in mammalian cells (e.g HPRT) and cytogenetic toxicity study – micronucleus test required according to in annex XIII section 8.4.2 and 8.4.3 were waived in accordance with Annex XI part 2. The studies are technically not feasible as the solubility of the substance in salt solution or in cell culture medium used in any cell culture experiments is extremely low (experimentally tested and summarized in a document attached in the IUCLID section 13). Consequently, the test substance is not expected to induce mutagenic effects in humans.

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:

1987-06-24 until 1987-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair substitution (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537).

Species / strain / cell type:

other: S. typhimurium TA 1538, TA 1535, TA 1537, TA 97, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (from rats induced with phenobarbital/ß-naphthoflavone)

Test concentrations with justification for top dose:

For the plate incorporation test: 10 - 1000 µg/plate
For the preincubation test: 5 - 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hot water (80°C).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

TA98, TA1538

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICI 191

Remarks:

TA 1537 and TA 97

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA)

Remarks:

used with all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- experiment I: plate incorporation test
- experiment II: pre-incubation test

DURATION
- Pre-incubation period: 30 min at 37°C (pre-incubation test)
- Expression time (cells in growth medium): 48 hours
- Number of independent experiments: 2 main experiments
- Number of replicates: 4 plates per concentration

Evaluation criteria:

The test substance was considered mutagenic if it induces at least a doubling of the number of spontaneous revertants and a dose response was recognizable. Clearly this rule of thumb has a questionable scientific foundation.

Statistics:

Mean values and standard deviations

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxic effects were visible at the highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test compound was dissolved in H2O at 80°C. Upon addition to the aqueous medium in the preincubation assay increasingly milky suspensions were formed starting at 50 µg/plate of the test substance.

Conclusions:

The test substance induced no genetic damage (considered not mutagenic) in the Ames test under the described experimental conditions.

Executive summary:

Potassium hexadecylphosphate was examined for mutagenic activity in the Ames test (plate incorporation and preincubation assay). The concentration range from 10 to 1000 µg/plate in the plate incorporation assay and from 5 to 500 µg/plate in the preincubation assay was tested with seven tester strains (TA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100, TA 102). Toxic effects were visible at the highest concentrations. The tests were performed in absence as well as in presence of a phenobarbital/ß-naphthoflavone-induced rat liver homogenate fraction (S9).

The test compound was dissolved in H2O at 80°C. Upon addition to the aqueous medium in the preincubation assay increasingly milky suspensions were formed starting at 50 µg/plate of the test substance.

The test substance did not induce an increase of the frequency of revertant colonies in any of the seven tester strains used. Responsiveness of the tester strains and activity of the S9 mix were demonstrated by using appropriate positive controls for each specific strain.

Thus, it can be concluded that neither the test substance per se, nor one of its metabolites formed under the described experimental conditions induced genetic damage in the Ames test and in the preincubation modification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

In the key study (performed with both plate incorporation and pre-incubation) as well as in the supporting study (performed only with plate incorporation) the test substance was proved to be not mutagenic in bacteria. In the key study, the test substance was investigated in the presence and absence of metabolic activation. Therefore Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100, and TA 102 were used. A pre-incubation assay as well as a plate incorporation assay were done independently. Concurrently negative and positive controls were tested to show the sensitivity of the test system. Hot water was used as vehicle. For each concentration applied 4 replicates were incubated. The following concentrations were used: 10-1000 µg/plate (plate incorporation assay) and 5-500 µg/plate (pre-incubation assay).

Toxic effects were seen at the highest concentration: In the plate incorporation assay 500 µg/plate (TA1538 –S9) and 1000 µg/plate (TA 1537 –S9, Ta 1538 –S9) produced reduced background growth. In the pre-incubation assay 50 µg/plate (TA 1537 –S9, TA 1538 -S9, TA 98 –S9 and TA 100 –S9, TA 102 –S9), 100 µg/plate (TA 1537 –S9, TA 1538 -S9, TA 98 –S9 and TA 100 –S9, TA 102 –S9) and 500 µg/plate (TA 1537 –S9, TA 1538 +/-S9, TA 98 –S9 and TA 100 –S9, TA 102 –S9) were cytotoxic sometimes disabling colony counting. Upon addition to the aqueous medium concentrations of 50 µg/plate and above milky suspensions were seen in the pre-incubation assay.

The experiments gave no indication that the test substance caused an increase in the revertant rate in either tester strain used. The positive controls gave expected results verifying the sensitivity of the test system.

In conclusion, the test substance is not mutagenic in the Ames test under the described conditions.

Gene mutation toxicity study in mammalian cells (e.g HPRT) and cytogenetic toxicity study - micronucleus test

Performance of an in vitro gene mutation study in mammalian cells (e.g HPRT) and cytogenetic toxicity study – micronucleus test with the test substance potassium hexadecyl hydrogen phosphate as required in REACH Regulation (EC) No. 19072006 Annex VIII section 8.4.3 are waived according to Annex XI part 2. Performance of the studies is technically not feasible as the test substance is not soluble in salt solutions e.g cell culture medium used in genetic toxicity studies with mammalian cells. Moreover, the solubility of the test substance in the relevant medium was experimentally tested and the results are summarized in a document attached in the IUCLID in section 13. The purpose of this document was to summarize the results of a solubility trial performed with the test substance as pre-experiment for genetic toxicity studies where cell culture medium is used. Based on the experimental results, evidence is presented to argue that due to extremely limited solubility of the test substance in cell culture medium, any cell culture experiments would not be valid and thus are scientifically unjustified.

Justification for classification or non-classification

Based on the available results and considerations potassium hexadecyl hydrogen phosphate was not classified and labelled for genotoxicity according to Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.