Phosphoric acid, mono- and di-C11-14 (linear and branched) alkyl esters

Endpoint summary

Additional information

Justification for read-across:

Reliable, relevant and adequate data on genotoxicity are available for the read-across substances Phosphoric acid, C9-15-branched and linear alkyl esters, potassium salts, Phosphoric acid, dodecyl ester, sodium and potassium salts and Dibutyl hydrogen phosphate.

The read-across approach is appropriate due to similar composition of source substances and registered substance.

Phosphoric acid alkyl esters are hydrolysed unspecifically by phosphatases, e.g. Acid phosphatase or Alkaline phosphatase. Both enzymes are found in most organisms from bacteria to human. Alkaline phosphatases are present in all tissues, but are particularly concentrated in liver, kidney, bile duct, bone and placenta. In human and most other mammals three isoenzymes of Alkaline phosphatase exist: intestinal ALP, placental ALP, tissue non-specific ALP (present in bone, liver, kidney, skin).

Seven different forms of Acid phosphatase are known in humans and other mammals. These are also present in different tissues and organs (predominantly erythrocytes, liver, placenta, prostate, lung, pancreas).

Linear and branched primary aliphatic alcohols are oxidised to the corresponding carboxylic acid, with the corresponding aldehyde as a transient intermediate. The carboxylic acids are further degraded via acyl-CoA intermediates in by the mitochondrial beta-oxidation process. Branched aliphatic chains can be degraded via alpha- or omega-oxidation (see common text book on biochemistry).

“The long chain aliphatic carboxylic acids are efficiently eliminated and aliphatic alcohols are therefore not expected to have a tissue retention or bioaccumulation potential.

Longer chained aliphatic alcohols within this category may enter common lipid biosynthesis pathways and will be indistinguishable from the lipids derived from other sources (including dietary glycerides).

A comparison of the linear and branched aliphatic alcohols shows a high degree of similarity in biotransformation. For both sub-categories the first step of the biotransformation consists of an oxidation of the alcohol to the corresponding carboxylic acids, followed by a stepwise elimination of C2 units in the mitochondrialβ-oxidation process. The metabolic breakdown for both the linear and mono-branched alcohols is highly efficient and involves processes for both sub-groups of alcohols. The presence of a side chain does not terminate the β-oxidation process, however in some cases a single Carbon unit is removed before the C2 elimination can proceed.” (OECD SIDS, 2006).

 

Assessment per endpoint:

a)           Gene Mutation Tests

Reverse gene mutation assay in bacteria

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to Phosphoric acid, C11-14-isoalkyl esters, C13-rich, CAS 154518-38-4 (100%) in DMSO at concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (Plate incorporation test and WP2uvrA without S9 mix and all strains with S9 mix), 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate (Pre-incubation test and Salmonella strains without S9 mix).

The assay was performed in two independent experiments both with and without liver microsomal activation. The plates incubated with the test item showed reduced background growth in all strains. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Mammalian cell gene mutation assay (hprt-test)

The test item Phosphoric acid, C11-14-isoalkyl esters, C13-rich was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in three independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The experimental part without metabolic activation was pre-maturely terminated based on exceedingly severe cytotoxic effects already at low concentrations. This experimental part was repeated as experiment IIA using lower concentrations. The data generated in experiment IIA are reported under experiment II without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without metabolic activation: 0.8, 1.6, 3.1, 6.3 µg/mL

with metabolic activation: 3.1, 6.3, 12.5, 25.0, 50.0 µg/mL

Experiment II:

without metabolic activation: 3.1, 6.3, 12.5, 25.0, 50.0, 75.0 µg/mL

with metabolic activation: 6.3, 12.5, 25.0, 50.0 µg/mL

No precipitation, visible to the unaided eye, was noted at analysable concentration levels in both main experiments.

The test item induced cytotoxicity showed a rather unusual course with little or no cytotoxicity up to the highest analysable concentration. At the next higher concentration virtually no cells survived even though the spacing between the concentrations was two fold or lower. In the first experiment the highest analysable concentration was 50.0 µg/mL with and 6.3 µg/mL without metabolic activation showing no cytotoxicity. The next higher concentrations of 75.0 µg/mL with and 12.5 µg/mL without metabolic activation resulted in a total kill of the cells. In the second experiment moderate cytotoxic effects leading to relative cell densities below 50% of the solvent control in both parallel cultures occurred at the maximum analysable concentration of 75.0 µg/mL without metabolic activation. In the presence of metabolic activation no cytotoxicity was noted at the maximum analysable concentration of 50.0 µg/mL. Again, a total kill of the cells occurred at the next higher concentration of 100.0 µg/mL without and 62.5 µg/mL with metabolic activation. The latter example of no relevant cytotoxicity at 50.0 µg/mL and a total kill of the cells at 62.5 µg/mL showed that the cytotoxicity occurred as a step rather than a gradient versus increasing concentrations.

No relevant and reproducible increase in mutant colony numbers/1E06 cells was observed in the main experiments up to the maximum analysable concentration. The mutant frequency did not exceed the historical range of solvent controls and the threshold of three times the mutation frequency of the corresponding solvent control was not reached or exceeded.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the second experiment without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above and the trend was not reproduced in the parallel culture under identical experimental conditions. Another trend was indicated in the second culture of the first experiment without metabolic activation. This trend however, is reciprocal, going down versus increasing concentrations and consequently without any biological relevance.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.6 up to 35.6 mutants per 1E06 cells; the range of the groups treated with the test item was from 7.9 up to 39.9 mutants per 1E06 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

b)           Chromosome Mutation Tests

In-vitro chromosome aberration tests (structural chromosomal aberration)

Four in-vitro tests for identification of structural chromosomal aberrations (similar or comparable to OECD Guideline 473) are available for different structurally related substances. The results of all available studies are evaluated in a Weight-of-evidence approach together with in-vivo data.

 

In a mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, adopted 21 July 1997, Chinese hamster lung fibroblast cell cultures were exposed to Phosphoric acid, C9-15-branched and linear alkyl esters, potassium salts (34.4% a.i., concentrations corrected for purity) in saline at the following concentrations:

6 h treatment

Without S9 mix: 0, 0.0781, 0.156, 0.313, 0.625, 1.25 mg/mL

With S9 mix: 0.526, 0.625, 0.743, 0.884, 1.05 mg/mL

24 h treatment (without S9 mix)

0, 0.0391, 0.0781, 0.156, 0.313, 0.625 mg/mL

48 h treatment (without S9 mix)

A: 0, 0.00488, 0.00977, 0.0195, 0.0391, 0.0781 mg/mL

B: 0, 0.0391, 0.0552, 0.0781, 0.110, 0.156 mg/mL

Phosphoric acid, C9-15-branched and linear alkyl esters, potassium salts was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. 

The test substance did not induce structural chromosome aberrations over background in all concentrations tested at any time point. However, the test substance induced numerical chromosome aberrations (polyploidy) after 48 h exposure without metabolic activation (this result will be discussed separately).

 

In a second mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, Chinese hamster lung cells (CHL/IU) were exposed to Phosphoric acid, dodecyl ester, sodium salt, in 0.5% sodium carboxymethyl cellulose solution at the following concentrations:

6-18 h treatment

Without S9 mix: 0, 23, 45, 90, 180 and 360 μg/mL

With S9 mix: 0, 23, 45, 90, 180 and 360 μg/mL

24 h treatment (without S9 mix): 0, 11, 23, 45, 90 and 180 μg/mL

48 h treatment (without S9 mix): 0, 11, 23, 45, 90 and 180 μg/mL

Sodium carboxymethyl cellulose (5% solution) was used as a vehicle, because the substance could not be dissolved in any other solvent, which is recommended for this study.

In a pretest, the test substance was tested up to 2880 µg/mL corresponding to 10 mM concentration. Substance adhering to the bottom of the test plates was observed at concentrations > 360 µg/mL in the group of 6 hours exposure +18 h recovery, and at concentrations > 180 µg/mL in the 24 hr and 48 hr treatment groups. Due to these precipitates, the exact 50% cell growth inhibitory concentration could not be reliably calculated.

In the main test, the substance was not tested up to cytotoxic concentrations because otherwise precipitation of the test substance impaired the analysis. Positive controls induced the appropriate response. 

The test substance did not induce structural chromosome aberrations over background in all concentrations tested at any time point. However, increase in the numerical chromosomal aberration frequency (polyploidy) was observed following continuous treatment for 24 and 48 hours without metabolic activation (this result will be discussed separately).

 

There are two further tests available, showing negative results for structural as well as numerical chromosomal aberrations:

In a mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, Chinese hamster V79 cells were exposed to Phosphoric acid, dodecyl ester, potassium salt in cell culture medium in the following concentrations:

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation.

Experiment I:

4 hours treatment

without metabolic activation: 15.6, 250, 500 and 1000 µg/mL

with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL

Experiment II:

4 hours treatment with metabolic activation: 900, 1600 and 1800 µg/mL

20 h treatment without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL

Precipitation of the test item was observed with and without metabolic activation in both experiments.

Toxic effects of the test item were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test item were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. The positive controls induced the appropriate responses.

 

In mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, Chinese hamster lung cells (CHL/IU) were exposed to Dibutyl hydrogen phosphate, in acetone at the following concentrations:

6-18 h treatment

Without S9 mix: 0, 0.10, 0.21, 0.41 mg/ml

With S9 mix: 0, 0.14, 0.27, 0.54 mg/ml

24 h and 48 h treatments (without S9 mix): 0, 0.06, 0.12, 0.24 mg/ml

Dibutyl hydrogen phosphate was tested up to cytotoxic concentrations as determined in a pretest.

In all experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation.

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. The positive controls induced the appropriate responses for structural chromosomal aberrations.

 

c)           Test for Aneugenic Effects (Polyploidy)

Test substance cell line	Test concentrations (cytotoxic concentrations underlined)	Cytotoxicity	Test results: Polyploidy
Phosphoric acid, C9-15-branched and linear alkyl esters, potassium salts CHL	6 h (- S9): 0, 0.0781, 0.156, 0.313, 0.625, 1.25 mg/mL 6 h (+ S9): 0.526, 0.625, 0.743, 0.884, 1.05 mg/mL 24 h (- S9): 0, 0.0391, 0.0781, 0.156, 0.313, 0.625 mg/mL 48 h (- S9) A: 0, 0.00488, 0.00977, 0.0195, 0.0391, 0.0781 mg/mL B: 0, 0.0391, 0.0552, 0.0781, 0.110, 0.156 mg/mL	48 h (- S9) A: 0.0781 mg/mL (growth rate 30%)   B: 0.0781, 0.110, 0.156 mg/mL (growth rates: 38, 19, 22 %)	Positive only at clearly cytotoxic concentrations 48 h (- S9) A: 0.0781 mg/mL (21.3 % polyploid cells) B: 0.0781, 0.110, 0.156 mg/mL (26.7, 24.2 % polyploidy cells, 0.156 mg/mL not analysable due to overt cytotoxicity)
Phosphoric acid, dodecyl ester, sodium salt CHL/IU	6-18 h (- S9): 0, 23, 45, 90, 180 and 360 μg/mL 6-18 h (+ S9): 0, 23, 45, 90, 180 and 360 μg/mL 24 h (- S9): 0, 11, 23, 45, 90 and 180 μg/mL 48 h (- S9): 0, 11, 23, 45, 90 and 180 μg/mL	6-18 h (- S9): not up to 2880 µg/mL 6-18 h (+ S9): > 1440 µg/L 24 h (- S9): > 1440 µg/L 48 h (- S9): > 720 µg/L	Positive 24 h (- S9): 45, 90 and 180 μg/mL (6.0[1], 12.5 and 12.5 % polyploid cells) 48 h (+ S9): 45, 90 and 180 μg/mL (26.0, 52.5 and 43.5 % polyploid cells)
Phosphoric acid, dodecyl ester, potassium salt V79 cells	Experiment I: 4 h (- S9): 15.6, 250, 500 and 1000 µg/mL 4 h (+ S9): 15.6, 500, 1000 and 1500 µg/mL Experiment II: 4 h (+ S9): 900, 1600 and 1800 µg/mL 20 h (- S9): 15.6, 31.3, 62.5 and 125.0 µg/mL	Experiment I: 4 h (- S9): 500 and 1000 µg/mL 4 h (+ S9): 1000 and 1500 µg/mL Experiment II: 4 h (+ S9): 1600 and 1800 µg/mL 20 h (- S9): 62.5 and 125.0 µg/mL	negative
Dibutyl hydrogen phosphate CHL/IU	6-18 h (- S9): 0, 0.10, 0.21, 0.41 mg/mL 6-18 h (+ S9): 0, 0.14, 0.27, 0.54 mg/mL 24 h, 48 h (- S9): 0, 0.06, 0.12, 0.24 mg/mL	6-18 h (- S9): 0.41 mg/mL 6-18 h (+ S9): 0.54 mg/mL 24 h, 48 h (- S9): 0.24 mg/mL	negative

[1]Ambiguous result as clearly positive results only defined as 10% or more

Possible aneugenic effects (induction of numerical chromosomal aberrations over background) have been detected in two of the four in-vitro chromosomal aberration tests.

 

In the first mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, adopted 21 July 1997, Chinese hamster lung fibroblast cell cultures were exposed to Phosphoric acid, C9-15-branched and linear alkyl esters, potassium salts.

Numerical chromosome aberrations (polyploidy) have been induced by the test substance after 48 h continuous treatment without S9 mix in the highest tested concentration of 0.0781 mg/mL. At this concentration 21.3 % of the cells showed numerical aberrations. The growth rate of the cells was reduced to 30% showing obvious cytotoxicity.

In a confirmatory test with 48 h continuous treatment without S9 mix at the concentration of 0.0781 mg/mL, 26.7 % of the cells showed numerical aberrations with a relative growth rate being reduced to 38%. At a concentration of 0.110 mg/mL 24.2 % of the cells showed numerical aberrations with the relative growth rate being reduced to 19%. The next higher dose of 0.156 mg/mL tested in the confirmatory test could not be evaluated due to severe cytotoxicity.

 

In the second mammalian cell cytogenetics assay (chromosome aberration test) according to OECD guideline 473, Chinese hamster lung cells (CHL/IU) were exposed to Phosphoric acid, dodecyl ester, sodium salt.

Numerical chromosome aberrations (polyploidy) have been induced by the test substance after 24 and 48 h continuous treatment without S9 mix.

Following the 24 h treatment without S9, at concentrations of 45, 90 and 180 µg/mL the numbers of polyploidy cells was increased to 6.0, 12.5 and 12.5%. In cells treated with the test substance for 48 h without metabolic activation, at concentrations of 45, 90 and 180 µg/mL the number of polyploidy cells was increased to 26.0, 52.5 and 43.5%.

Positive controls induced the appropriate response only for structural chromosomal aberrations. 

In a pretest, the test substance was tested up to 2880 µg/mL corresponding to 10 mM concentration. Substance adhering to the bottom of the test plates was observed at concentrations > 360 µg/mL in the group of 6 hours exposure +18 h recovery, and at concentrations > 180 µg/mL in the 24 hr and 48 hr treatment groups. Due to these precipitates, the exact 50% cell growth inhibitory concentration could not be reliably calculated.

The test substance was not cytotoxic up to the highest dose of 2880 µg/mL after 6 h exposure without S9. After short-term exposure with S9, there was no cytotoxicity up to 1440 µg/mL but the relative cell growth dropped to ca. 10% at the next higher dose level of 2880 µg/mL. In the contrary, doses between 90 and 180 µg/mL rather stimulated cell growth up to 160 %. Between 180 and 1440 mg/mL this plateau was constant for the 6 h exposure with and without metabolic activation.

In the tests with continuous treatment without S9, stimulation of cell growth up to 150% was again observed between 90 and 1440 mg/mL after 24 h exposure. At 2880 mg/mL the substance was cytotoxic with a cell growth of approximately 20%. After 48 h exposure, the cell growth was stimulated between 180 and 720 µg/mL with cytotoxicity at and above 1440 µg/mL.

In the main test, the substance was not tested up to cytotoxic concentrations because otherwise precipitation of the test substance impaired the analysis.

In the english translation of the report summary the following information was given: "No increased incidence of structural aberration and numerical aberration (polyploidy) was observed in the metabolic activation of short-term treatment method. Although the incidence of structural aberration did not increase in the non-metabolic activation, a slightly increased incidence of numerical aberration (polyploidy) was observed. On the other hand, although there was no incidence of structural aberration after both of 24 h and 48 h treatments in the continuous treatment method, a significant concentration-dependent increase in numerical aberration (polyploidy) was observed. In addition to many octoploid cells, polyploid cells with pulverisation or premature chromosome condensation were also observed. The mechanism that induced the increase in polyploidy was unclear. However, considering that pulverisation and premature chromosome condensation might be induced by inner factors when multinucleated or micronucleated cells generated by cell fusion, inhibition of cytokinesis, spindle fiber inhibition, chromosome bridge, and chromosome fragment, unresponsively undergo cell division and that endoreduplication was not observed, it is estimated that the increased polyploidy was induced by the effects of the test substances on cell fusion and cytokinesis."

 

Comments on reliability / validity of the test:

Only a short English translation was available for this test. Although it is a GLP test, we have some doubts on the results due to following facts:

The high degree of precipitation might have impaired the test, as it impaired the exact evaluation of the cytotoxicity.

The kind of growth curve seen in this test was not observed in any other of these tests, neither with the same cell type nor with the C12, K-salt in V79 cells (see attachment).

In addition, two chromosome aberration tests available for Phosphoric acid, dodecyl ester, potassium salt in Chinese hamster V79 cells and Dibutyl hydrogen phosphate Chinese hamster lung cells were clearly negative for polyploidy even when tested up to cytotoxic concentrations.

 

Mammalian Erythrocyte Micronucleus Test

A negative in vivo micronucleus study is available for a substance closely related to the branched AP.

 

Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts was tested in an in vivo micronucleus test with male Crl: CD(SD) rats (8 weeks old at dosing) to examine its ability to induce micronucleated cells in the bone marrow.

The animals (5 animals per group) received oral gavage administration of the negative control (water for injection), the test substance at 250, 500, 1000 or 2000 mg/kg or the positive control (cyclophosphamide monohydrate 20mg/kg) twice at a 24 hr interval. Bone marrow cells were collected 24 hr after the final administration. Bone marrow cell specimens were prepared to examine the incidence of micronucleated immature erythrocytes (MNIMEs) and percentage of immature erythrocytes (IMEs).

No statistically significant difference was detected in the numbers of MNIMEs per 10000 IMEs (2000 cells/animal x 5 animals/group) between the test substance groups and the negative control group. No significant decrease was detected in the percentage of IMEs in any of the test substance groups compared to the negative control group, indicating no bone marrow toxicity. In contrast, the number of MNIMEs signincantly increased, and the percentage of IMEs significantly decreased in the positive control group compared to the negative control group.

The incidences of MNIMEs in the negative control groups were within the range of the historical control data of the testing facility. Significant increases were detected in the incidence of MNIMEs in the positive control group. These results confirmed the validity of this study.

It was concluded that Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts did not induce micronucleated erythrocytes in the rat bone marrow cells under the conditions employed in this study.

Additional supporting information on oral absorption

Phosphoric acid alkyl esters are expected to undergo hydrolysis to aliphatic alcohols and Phosphoric acid in the gastro-intestinal tract by intestinal phosphatases. Thus, the gastro-intestinal absorption of Phosphoric acid alkyl esters is assumed to play a minor role compared to the absorption of the metabolites aliphatic alcohols and Phosphoric acid.

This is supported by data collected by the Dutch Committee on Updating of Occupational Exposure Limits on Tributyl Phosphate (Health Council of the Netherlands, 2005): “After administration of oral doses of tributyl phosphate of 156 mg/kg bw, the parent compound was detected in the gastrointestinal tract, the blood, and the liver within 30 to 60 minutes. Following a single dose, the highest amount was found in the gastrointestinal tract (not quantified), and 5.7% of the dose was detected in the other tissues (no further information given).”

 

Long chain aliphatic alcohols can be expected to be orally absorbed depending on their chain-length. As stated in the OECD SIDS Initial Assessment Report on long chain alcohols, aliphatic alcohols “orally administered aliphatic alcohols […] show a chain-length dependant potential for gastro-intestinal absorption, with shorter chain aliphatic alcohols having a higher absorption potential than longer chain alcohols.” (OECD SIDS, 2006)

 

Phosphate is an essential nutrient which is absorbed in the small intestine via passive diffusion (paracellular transport) as well as via active transport by sodium-dependent phosphate co-transporters (Sabbagh et al, 2011).

 “Net absorption from a mixed diet has been reported to vary between 55-70% in adults (Lemann 1996; Nordin, 1986) and between 65-90% in infants and children (Ziegler and Fomon, 1983). There is no evidence that, contrary to calcium, absorption efficiency varies with dietary intake. Phosphate absorption is greatest in jejunum and takes place by a saturable, active transport mechanism, facilitated by 1,25-dihydroxyvitamin D, as well as by passive diffusion (Chen et al., 1974)” (EFSA, 2005).

Discussion of the relevance of the numerical chromosome aberrations found in-vitro:

In one test, polyploidy was only detected in the range of clear cytotoxicity. The second test showed a high percentage of polyploidy cells without a clear indication of cytotoxicity. This test was only available as a short abstract and showed some major deficiencies leading to a restricted validity. Nevertheless, as it cannot be completely excluded that the substance indeed has some capacity to induce polyploidy, the biological relevance of the finding will be discussed:

 

The numerical chromosomal aberrations have been detected in tests performed according to OECD test guideline 473. In the introduction of this test guideline, it is explicitly mentioned that: “The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. … An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. However, this guideline is not designed to measure numerical aberrations and is not routinely used for that purpose” (OECD Guideline 473).

Polyploidy is a common finding in chromosome aberration assays in vitro. Although aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and can simply indicate cell cycle perturbation; it is also commonly associated with increasing cytotoxicity. If polyploidy, but no structural chromosome breakage, is seen in an in vitro assay, generally a negative in vivo micronucleus assay with assurance of appropriate exposure would provide sufficient assurance of lack of potential for aneuploidy induction (CDER / CBER 2012).

 

Evaluation of published and unpublished data by ECETOC has shown that induction of polyploidy can be used as an indicator of aneuploidy. It could also be shown that the frequency of chemicals that induce polyploidy in the absence of other genotoxic events is low and the biological relevance

of the chemicals that only induced polyploidy is largely unknown (Aardema M.J. 1998). For the substance to be registered, in all experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test items with and without metabolic activation. In addition it was shown that the substance was negative in bacterial and mammalian cell gene mutation assays.

 

Possible causes and cellular targets for the induction of polyploidy can be similar to those cited for aneuploidy, namely effects on the spindle apparatus (kinetochores, centrioles and microtubules and associated proteins) and effects on membrane components. However, unlike aneuploidy, polyploidy is never caused by DNA damage and can arise by additional mechanisms such as failure of cytokinesis and formation of a restitution nucleus, nuclear fusion within a binucleate cell and also the uncoupling of cell division and DNA replication giving endoreduplication or endomitosis (Mitchell G. 1995).

The main mechanisms leading to polyploidyare mitotic slippage or endomitosis and thus so called epigenetic mechanisms. Both mechanisms are clearly following a threshold dose-response curve. The existence of biologically meaningful threshold dose–response curves for events is probable in the case of mechanisms which require the involvement of more than one target. Elhajouji et al. showed that classic spindle poisons (nocodazole, colchicine, carbendazim and mebendazole) induced chromosome loss in a threshold concentration–response relationship with a no-effect level.

 

Assessing the genotoxic potential of other surfactants with a similar general structure (alcohol sulphates) HERA (2002) has concluded that: “Based on the chemical structures of the category surfactants, no specific affinity for, or reactivity with, chromosomal DNA or structural proteins is anticipated. It has been suggested in the literature that cellular toxicity correlates with lipophilicity, and that lipophilic materials that are cytotoxic are more likely to show positive results inin vivoassays for genotoxicity based simply on generalized cytotoxic effects (Rosenkranz and Klopman, 1983). If this is the case, chromosomal damage in response to high surfactant doses, if observed, would likely be secondary to surfactant-induced cytotoxicity and a generalized disruption of cellular integrity. […] However, since metabolism of the surfactants curtails and destroys their surfactant properties, cytogenetic effects secondary to generalized cytotoxicity should be limited, unless the dose or mode of administration is such that the metabolic capacity of the test animals is overwhelmed. The oral, repeated dose studies conducted at the MTD indicate that such conditions are not likely to be achieved in a properly conducted study. Therefore, since the surfactant structures are not expected to modify genetic macromolecules, and the materials are rapidly metabolized to short-chained, polar compounds in mammals, direct or indirectin vivocytogenetic effects are not expected.” This may also be relevant for the Phosphoric acid alkyl esters.

 

In both in vitro tests that showed positive results for polyploidy, the need for prolonged exposure (24 or 48 h without metabolic activation) suggests that a short exposure, albeit at high concentration, is unlikely to be a hazard for animals or humans. There is evidence of an in vitro threshold at approximately 45 µg/mL. Microsomal metabolism (S9 mix) virtually eliminates PAE-inducedpolyploidy. Taking into consideration the rapid metabolism of Phosphoric acid alkyl esters, in vivo concentrations of the substance itself in the range of 45 µg/mL over longer periods (24 h and more) are unlikely to be achieved. This implies that the low-level systemic exposure of the test substance itself after absorption may well not be hazardous.

Dermal absorption of Phosphoric acid alkyl esters can be assumed to be <10%. Estimated dermal penetration rates were in the range of5.20E-06 to 1.04E-03 mg/cm²/h (0.0052 to 1.04 µg/cm²/h). Tissue concentrations of 45 µg/mL are highly unlikely to be achieved by dermal exposure toPhosphoric acid alkyl esters.

The effect is further impacted by pharmacokinetic factors, since the binding to the target receptor is usually reversible and repeated exposure particularly at low doses may not be additive.

 

Supporting information about metabolites:

Phosphoric acid alkyl esters are hydrolysed unspecifically by phosphatases, e.g. acid phosphatase or alkaline phosphatase. Both enzymes are found in most organisms from bacteria to human. Alkaline phosphatases are present in all tissues, but are particularly concentrated in liver, kidney, bile duct, bone, placenta. In human and most other mammals three isoenzymes of Alkaline phosphatase exist: intestinal ALP, placental ALP, tissue non-specific ALP (present in bone, liver, kidney, skin).

Seven different forms of Acid phosphatase are known in humans and other mammals. These are also present in different tissues and organs (predominantly erythrocytes, liver, placenta, prostate, lung, pancreas).

 

Phosphate as such is not metabolised. It “is an essential dietary constituent, involved in numerous physiological processes, such as the cell’s energy cycle (high-energy pyrophosphate bonds in adenosine triphosphate [ATP]), regulation of the whole body acid-base balance, as component of the cell structure (as phospholipids) and of nucleotides and nucleic acids in DNA and RNA, in cell regulation and signalling by phosphorylation of catalytic proteins and as second messenger (cAMP). Another important function is in the mineralization of bones and teeth (as part of the hydroxyapatite)” (EFSA, 2005).

 

Linear and branched primary aliphatic alcohols are oxidised to the corresponding carboxylic acid, with the corresponding aldehyde as a transient intermediate. The carboxylic acids are further degraded via acyl-CoA intermediates in by the mitochondrial beta-oxidation process. Branched aliphatic chains can be degraded via alpha- or omega-oxidation (see common text book on biochemistry).

“The long chain aliphatic carboxylic acids are efficiently eliminated and aliphatic alcohols are therefore not expected to have a tissue retention or bioaccumulation potential (Bevan, 2001). Longer chained aliphatic alcohols within this category may enter common lipid biosynthesis pathways and will be indistinguishable from the lipids derived from other sources (including dietary glycerides) (Kabir, 1993; 1995a,b). A comparison of the linear and branched aliphatic alcohols shows a high degree of similarity in biotransformation. For both sub-categories the first step of the biotransformation consists of an oxidation of the alcohol to the corresponding carboxylic acids, followed by a stepwise elimination of C2 units in the mitochondrial β-oxidation process. The metabolic breakdown for both the linear and mono-branched alcohols is highly efficient and involves processes for both sub-groups of alcohols. The presence of a side chain does not terminate the β-oxidation process, however in some cases a single Carbon unit is removed before the C2 elimination can proceed.” (OECD SIDS, 2006)

 

The metabolites of Phosphoric acid alkyl esters enter normal metabolic pathways and are therefore indistinguishable from Phosphate and lipids from other sources.

Phosphates were furthermore negative in a number of tests for genotoxicity in vitro and in vivo (summarised in JECFA, 1982). Phosphoric acid was negative in the in vitro tests undertaken (ECHA, 2011a). Studies on the carcinogenicity of phosphates have not been identified. However, in view of the physiological roles of phosphate in the body, it is unlikely that oral exposure to phosphoric acid would pose a carcinogenic risk.

The weight of evidence is further supported by the fact that the in vivo micronucleus assay with Phosphoric acid, C13-15 branched and linear alkyl esters, potassium salts gave a negative result.

We conclude that Phosphoric acid, mono- and di- C11-14 (linear and branched) alkyl esters does not pose a significant potential hazard for humans.

The Ames test, the Mammalian cell gene mutation assay and the In vivo mammalian erythrocyte micronucleus test showed negative results. There is no reason to believe that the negative results would not be relevant to humans.

 

 

References

M.J. Aardema, S. Albertini, P. Arni, L.M. Henderson, M. Kirsch-Volders, J.M. Mackay, A.M. Sarrif, D.A. Stringer, R.D.F. Taalman (1998) Aneuploidy: a report of an ECETOC task force. Mutation Research 410_1998.3–79

 

EFSA (2005) Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the Tolerable Upper Intake Level of Phosphorus, The EFSA Journal (2005) 233, 1-19; available via internet:http://www.efsa.europa.eu/en/efsajournal/doc/233.pdf

 

Elhajouji A., Van Hummelen P., Kirsch-Volders M. (1995) Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes, Environ. Mol. Mutagen. 26 292–304.

 

Health Council of the Netherlands: Committee on Updating of Occupational Exposure Limits. Tributyl phosphate; Health-based Reassessment of Administrative Occupational Exposure Limits. The Hague: Health Council of the Netherlands, 2005; 2000/15OSH/158; available via internet: http://www.gezondheidsraad.nl/sites/default/files/[e-mail]

 

HERA (2002)Human Health Risk Assessment: Alcohol Sulphates, available via internet:http://www.heraproject.com/RiskAssessment.cfm?SUBID=3

 

ICH (S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) June 2012 ICH):http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074931.pdf

 

Marzulli FN, Callahan JF, Brown DWC (1965) Chemical structure and skin penetrating capacity of a short series of organic phosphates and phosphoric acid.

 

Mitchell I de G., Lambert T.R., Burden M.,Sunderland J., Porter R.L. and Carlton J.B. (1995)Is polyploidy an important genotoxic lesion?Mutagenesis 10 (2): 79-83.

 

OECD SIDS, 2006 SIDS Initial Assessment Report for SIAM 22, Long Chain Alcohols (C6-22 primary aliphatic alcohols), available via internet:http://www.aciscience.org/docs/SIDS_LCA_tome2.pdf

 

OECD GUIDELINE 473 FOR THE TESTING OF CHEMICALS: In Vitro Mammalian Chromosome Aberration Test Adopted: 21st July 1997

 

Sigmon CF, Daugherty ML (1985) Chemical Hazard Information Profile, Draft Report, Tri(alkyl/alkoxy) Phosphates, September 23, 1985, Report of Oak Ridge National Laboratory to US EPA; available via internet:http://nepis.epa.gov/Adobe/PDF/91005XP6.PDF

 

ten Berge WF. (2009). A simple dermal absorption model: Derivation and application. Chemosphere 75, 1440-1445

 

Wilschut, A., ten Berge, W. F., Robinson, P. J., McKone, T. E. (1995) Estimating skin permeation. The Validation of five mathematical skin permeation models. Chemosphere 30, 1275-1296http://home.planet.nl/~wtberge/qsarperm.html

Justification for selection of genetic toxicity endpoint
No single key study has been selected. The conclusion is based on the following assays: Bacterial reverse mutation assay (Ames test); Mammalian cell gene mutation assay; in-vivo mammalian chromosome aberration test

Short description of key information:
Negative in all tests conducted:
- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471 and GLP); toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.
- Mammalian cell gene mutation assay with Chinese hamster lung fibroblasts (V79) (met. act.: with and without) (OECD Guideline 476 and GLP); cytotoxicity: yes
- In vitro chromosome aberration assays (OECD TG 473 and GLP); cytotoxicity: yes; negative for structural aberrations, however, induction of polyploidy observed in some of the available tests
- In vivo mammalian erythrocyte micronucleus test (met. act.: with and without) (OECD Guideline 474 and GLP); cytotoxicity: no

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Directive 67/548/EEC, Annex 1 and Regulation (EC) No. 1272/2008: Based on the available data, no classification is needed