Phosphoric acid, C16-18-alkyl esters, potassium salts

Endpoint summary

Description of key information

Subchronic (90-day) study oral (gavage), rat (Sprague-Dawley) m/f (OECD guideline 408, GLP): BMDL10: 240.30 mg/kg bw/d (corresponding BMD = 374.61 mg/kg bw/d) (both sexes); read-across substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts  Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test oral (gavage), rat (Sprague-Dawley) m/f (OECD guideline 422, GLP no data): NOAEL systemic toxicity: 1000 mg/kg bw/day (both sexes); read-across substance Phosphoric acid, dodecyl ester, sodium salt  Subacute (28-day) study oral (dietary), rat (Sprague-Dawley) m/f (OECD guideline 407, GLP): NOAEL (females) 1564 mg/kg bw/day (= NOEL males); read-across substance Mono-myristyl phosphate   Reliable data on repeated dose toxicity of Phosphoric acid esters (PAE) are available from 42-day and 90-day gavage studies as well as from a 28-day feeding study in rats on read-across substances (Phosphoric acid, dodecyl ester, sodium salt; Mono-myristyl phosphate; Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts).  The main effects observed were local irritative effects at the site of application (forestomach gastritis) seen in the gavage studies, focal corticomedullary mineralisation in the kidneys of female rats and hypertrophy of the cortical glomerular zone of the adrenal gland. The findings in the forestomach as well as in the kidneys are either judged as not substance related or as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans. Based on these data, the most critical effects were seen in the adrenal glands showing hypertrophy of the zona glomerulosa in the 90 d study. The BMDL10 is 240.30 mg/kg bw/d (corresponding BMD = 374.61 mg/kg bw/d).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL10

240.3 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

All available studies are high quality guideline studies with original RL1. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on read-across substances.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable data on repeated dose toxicity of Phosphoric acid esters (PAE) are available from 42-day and 90-day gavage studies as well as from a 28-day feeding study in rats on read-across substances (Phosphoric acid, dodecyl ester, sodium salt; Mono-myristyl phosphate; Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts): In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422 Phosphoric acid, dodecyl ester, sodium salt was administered 12 Sprague-Dawley rats/sex/dose daily by oral gavage at dose levels of 0 (control), 250, 500, 1000 mg/kg bw/day. The males were exposed 14 days before mating, through the mating period, up to 1 day before termination (42 days in total). The females were exposed 14 days before mating, through the mating and gestation period, up to day 4 of lactation (42 to 45 days in total). Additional 5 males and 5 females were added to the control group and 1000 mg/kg group as a recovery group to examine the reversibility of the effects. Those animals were observed for another 14 days. Administration of Phosphoric acid, dodecyl ester, sodium salt had no effect on any of the following: general condition, results of function tests, grip strength, spontaneous motor activity, body weight, food consumption, or the results of urinalysis (including water intake), or haematological tests.

A mild but significant increase of the serum ALT in males of the highest dose group at the end of the administration period was not accompanied by histopathological findings and was within the range of physiological variations in historical controls. Therefore it was judged, that these findings were not of toxicological relevance.

In the pathological examination performed at the end of the administration period, gross observation showed indentation of the forestomach in the animals of the 250 mg/kg and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg and higher dose groups. Histological observation showed erosions/ulcers of the forestomach, thickening of the forestomach mucosa, and edema in the submucosal tissue. In the pathological examination performed at the end of the recovery period, only one male animal of the highest dose group showed moderate thickening of the anterior stomach mucosa.

In a subacute toxicity study according to OECD 407, Mono-myristyl phosphate was administered for a period of 28 days to 5 Sprague-Dawley rats/sex/dose in diet, at dose levels of 0, 2250, 6000, 15000 ppm (0, 227, 505, 1564 mg/kg bw/day).

There were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross pathology. An increased incidence of focal corticomedullary mineralisation was observed in the kidneys of females treated with 6000 or 15000 ppm. Intratubular mineralisation occurs spontaneously in many laboratory animals, and is often observed in rats, where the incidence tends to be higher in females. It is most usually detected at the corticomedullary junction, and the consequences of mineralisation are normally minimal. There were no associated blood chemical changes indicative of renal dysfunction and the macroscopic changes were considered not to represent any adverse effect on the health of the animals.

In a subchronic toxicity study according to OECD guideline 408, Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts (34.35% a. i.) was administered to 5 Sprague-Dawley rats/sex/dose by oral gavage at dose levels of 0 (control), 8, 40, 200 and 1000 mg/kg bw/day). The animals were dosed for 91 days. Additional 5 males and 5 females were added to the control group and 1000 mg/kg group as a recovery group to examine the reversibility of the effects. Those animals were observed for another 14 days.

No adverse reaction was observed in detailed clinical observation, sensory response test, grip strength test, locomotor activity, ophthalmology, haematology, blood biochemistry or organ weight.

The changes observed during the dosing period or at the end of the dosing period such as salivation, abnormal respiratory sound, low body weight, low urine pH, thickening of the forestomach mucosa, dilatation of cecum, hyperplasia and erosion of squamous cells of the forestomach and hypertrophy of the cortical glomerular zone of the adrenal gland. In adrenal gland, mild hypertrophy of the cortical glomerular zone was observed in 3 males and 1 female at 200 mg/kg bw/d, mild to moderate hypertrophy was seen in 8 males and 9 females at 1000 mg/kg bw/d. There was no significant difference in the incidence at 1000 mg/kg bw/d between both sexes. The cells in the glomerular zone in the control group had fewer vacuoles, while in the hypertrophic glomerular zone, cytoplasm was pale-colored with vacuoles.

All effects observed during the dosing period were not observed during the recovery period or at the end of the recovery period except for the change in the adrenal gland. The change in the adrenal gland also became milder and these changes were confirmed to be reversible.

The above results show that toxic effect after repeated oral administration of Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts appears mainly in the adrenal gland and forestomach.

Justification for read-across:

The read-across approach is appropriate due to similar composition of source and registered substance. From the available data is can be concluded that the repeated dose toxicity of substances with different alkyl moieties (C12, C14, C9-15 linear and branched) is comparable.

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).

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; 1995 a,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 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 theβ-oxidation process, however in some cases a single Carbon unit is removed before the C2 elimination can proceed.” (OECD SIDS, 2006)

The PAEs with branched fatty alcohols can be considered as a worst case scenario because the metabolism of the resulting branched fatty acids occurs less efficient compared to linear fatty acids.

Taking all available studies into consideration, the main effects seen in these animal studies are local irritative effects at the site of application (forestomach gastritis) seen in gavage studies, focal corticomedullary mineralisation in the kidneys of female rats and hypertrophy of the cortical glomerular zone of the adrenal gland. The findings in the forestomach as well as in the kidneys are either judged as not substance related or as not relevant in view of a potential serious health risk for humans due to significant different anatomic situation and exposure probability in humans as explained below in detail.

Forestomach effects:

Forestomach findings related to an irritant activity of the test item are common findings in rat gavage studies. The stomach of rats is anatomically different from the human stomach. The rat stomach consists of two anatomically distinct parts of approximately equal size: a non-glandular forestomach and a glandular stomach. The forestomach is connected to the oesophagus at the gastro-oesophageal junction, and is clearly separated from the glandular stomach by a distinct border called the limiting ridge. The forestomach is not present in humans. The main function of the rat forestomach is storage and trituration of ingested food prior to digestion in the glandular stomach. The forestomach is a holding compartment and due to this function a long exposure time to orally - especially by gavage as bolus dose - administered doses in this organ occurs. Thus, an irritant test item can easily take effect at the forestomach squamous epithel.

A functional equivalent to the rat forestomach is missing in humans. A histological similar in humans is the oesophagus, because the epithelium of the human oesophagus is morphologically of the same type as the forestomach epithelium of rats. However, tissue exposure in the human oesophagus is likely to be minimal compared to tissue exposure in the rat forestomach since the epithelial contact is much shorter in the human oesophagus compared to the rat forestomach and it seems very unlikely that exposure to concentrations far below those having an irritating potential is hazardous to man. Further on, a constantly repeated oral bolus ingestion of substances like the PAEs, is very unlikely. When test items are administered with the feed or the drinking water – exposure procedures more realistic to the exposure situation of humans –, forestomach effects will be unlikely to occur.

This has also been proven for the PAE (mono-myristyl phosphate, C14 acid form) in a 28-day feeding study in rat with daily dose levels up to 1564 mg/kg bw and thus exceeding those used in this 90-day gavage study. Stomach effects were completely absent in this feeding study.

Moreover, the forestomach effects have been shown to be reversible: In the animals sacrificed at the end of the recovery period, no change was recognised in the forestomach (90-day study).

Therefore, forestomach findings in rat studies resulting from irritation associated with high-concentration gavage dosing as seen in gavage studies on PAE should not form the basis for determining systemic toxicity or quantitative systemic toxicity potency estimates. These local reversible effects are not judged as relevant in view of a potential serious health risk for humans.

Focal corticomedullary mineralisation in kindneys:

In the 28-d feeding study with mono-myristyl phosphate, C14 acid form, females treated with 15000 or 6000 ppm (1564 or 505 mg/kg bw/day) showed an increased incidence of focal corticomedullary mineralisation in the kidneys.

Intratubular mineralisation occurs spontaneously in many laboratory animals, and is often observed in rats, where the incidence tends to be higher in females. It is most usually detected at the corticomedullary junction, and the consequences of mineralisation are normally minimal. There were no associated blood chemical changes indicative of renal dysfunction and the macroscopic changes therefore were considered not to represent any adverse effect on the health of the animals.

In the 28-d study Na+, K+, H+ and Ca2+ were not affected, but serum phosphate levels were significantly higher in males of the highest dose group (15000 ppm) and slightly but not significantly increased in females of the two upper dose groups (6000 and 15000 ppm). However, individual phosphate levels were within the normal range for rats of this strain, according to the study authors.

According to EFSA (2005b), ectopic calcification as a result of high dietary phosphorus intake, as has been observed in mice and rats with normal kidney functions before exposure, has not been reported in humans with an adequate renal function. This might occur however in patients with end-stage renal disease associated with a variety of syndromes and (malignant) conditions. However, in these conditions, the hyperphosphatemia is not a direct, but a secondary effect.

It should also be noted that standard diets for laboratory animals generally have a relatively high phosphorus and low calcium content (JECFA, 1982). It cannot be excluded therefore that the observed effects in some of the animal studies were associated with the relatively low calcium intakes, rather than the high phosphorus intake as such. Besides the sensitivity of the PTH-vitamin D axis to variations in calcium and phosphorus intake might be different between animals and humans. This effects was not observed in the other available studies.

Adrenal gland hypertrophy:

In the 90-day study a mild hypertrophy of the cortical zona glomerulosa of the adrenal gland has been described for the 200 mg/kg bw/d (3 males, 1 female) and mild-to-moderate hypertrophy at 1000 mg/kg bw/d (8 males, 9 females) dose groups. In the enlarged glomerular zone, cytoplasm was pale-colored due to vacuoles, which were probably lipid. The change in the adrenal gland became milder during the recovery period showing a clear tendency for recovery and thus, the effects are considered to be reversible.

It is not clear, if this hypertrophy may be a physiological response to increased potassium intake as it has been reported in EFSA (2005a).

This interpretation is in line with the toxicity profiles of the potential metabolites:

The oral administration of 1000 mg PAE/kg bw/d will in the worst case (100% absorption) lead to a systemic phosphate dose of approximately 220 – 320 mg phosphate/kg bw/d.

EFSA elvaluated the toxicity of phosphorous as phosphate in 2005 (EFSA, 2005b).

“Adverse effects of excessive phosphorus intake, such as hyperphosphatemia, leading to secondary hyperparathyroidism, skeletal deformations, bone loss, and/or ectopic calcification have been reported in animal studies. However, such effects were not observed in studies in humans, except in patients with end stage renal disease. Although in acute or short term loading studies an increase in serum parathyroid hormone (PTH) levels has been found, no significant changes could be demonstrated in longer term studies with dosages up to 3000 mg/day (for 6 weeks). In these studies no evidence was found for effects on markers of bone remodelling and the Panel does not consider these to be adverse effects. Similarly, the Panel found no convincing evidence to support suggestions that high phosphorus diets would aggravate the effects of a state of secondary hyperparathyroidism induced by inadequate calcium intakes, or an inadequate vitamin D status.“

“The Panel concludes that the available data are not sufficient to establish an upper level for phosphorus.

The available data indicate that normal healthy individuals can tolerate phosphorus (phosphate) intakes up to at least 3000 mg/day without adverse systemic effects. In some individuals, however, mild gastrointestinal symptoms have been reported if exposed to supplemental intakes >750 mg phosphorus per day. There is no evidence of adverse effects associated with the current dietary intakes of phosphorus in EU countries.“

The repeated dose toxicity of long chain aliphatic alcohols, which would be the second metabolite, has been reviewed in a SIDS Initial Assessment Report (OECD SIDS, 2006). The authors concluded that the “repeat dose toxicity of the category of long chained alcohols with chain lengths ranging from C6 to C22 indicates a low order of toxicity upon repeated exposure. Typical NOAELs recorded for this category range between ca. 200 mg/kg/day to 1000 mg/kg/day in the rat upon sub-chronic administration via the diet. At the lower end, members of this category induce local irritation at the site of first contact. Other notable findings observed for several members within this group suggest mild changes consistent with low-grade liver effects with the changes in essentially linear alcohols being slightly more pronounced than in linear alcohols. Typical findings include: slightly increased liver weight, in some cases accompanied by clinical chemical changes but generally without concurrent histopathological effects. The significance of these effects for human health is not likely to be significant given that the aliphatic alcohols are efficiently metabolised and the biotransformation products are very similar or indistinguishable from those derived from common dietary sources (triglycerides) and that human intake from these dietary sources is likely to be considerably higher than the exposures from uses involving aliphatic alcohols.”

Based on the available data, the most critical effects were seen in the adrenal glands showing hypertrophy of the zona glomerulosa in the 200 and 1000 mg/kg bw/d dose groups of the 90 d study.

Due to the low incidence and mild expression of effects in the 200 mg/kg bw/d dose group as well as the distance between the dose groups the derivation of a benchmark dose instead of a NOAEL as proposed by the study authors seems more appropriate.

The details for BMDL10 derivation are described in the attached document “Benchmark dose derivation for repeated dose toxicity”.

The BMDL10 obtained for this study is 240.30 mg/kg bw/d (corresponding BMD = 374.61 mg/kg bw/d).

The hypertrophy of the zona glomerulosa in the adrenal gland observed in the oral 90-day study in rats is probably also relevant to humans. However, since moderate effects in a high were only observed in the highest dose group, and there was a clear tendency to reversibility in the recovery group, the effect does not give rise to concern, even though it is most probably an adverse effect and therefore is taken into account in risk assessment. There are no data gaps in repeated dose toxicity.

References

EFSA, 2005a: 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 Potassium, The EFSA Journal (2005) 193, 1-19, available via internet: http://www.efsa.europa.eu/en/efsajournal/doc/193.pdf

EFSA, 2005b: 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/de/scdocs/doc/233.pdf

OECD SIDS, 2006: SIDS Initial Assessment Report For SIAM 22: Long chain alcohols, available via internet: http://www.aciscience.org/docs/Draft_SIDS_Long_Chain_Alcohols_1.pdf

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The study with the longest duration and most critical effects has been selected as key study; for details see discussion.

Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: adrenal gland

Justification for classification or non-classification

In conclusion, the results of the available data on repeated dose toxicity indicate that Phosphoric acid, C16-18-alkyl esters, potassium salts does not need to be classified for repeated dose toxicity according to Directive 67/548/EEC as well as CLP, EU GHS (Regulation 1272/2008/EC) and therefore labelling is not necessary.