Reaction mass of hexadecyl dihydrogen phosphate and dihexadecyl hydrogen phosphate

Endpoint summary

Description of key information

The structural analogue, Phosphoric acid, tetradecyl ester, was administered daily in graduated doses to 3 groups of test animals. Animals of an additional control group were handled identically as the dose groups but received plain diet in this study. The 4 groups comprised 5 male and 5 female Wistar rats.
Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Phosphoric acid, tetradecyl ester, was considered to be 1564 mg/kg body weight/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Sept 1995 - Jan 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP conform guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd., Manston, Kent, U.K.
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 128 - 166g
- Housing: in groups of five by sex in polypropylene grid-floor cages
- Diet (e.g. ad libitum): Rat and Mouse SQC Expanded Diet No. 1 Ground (Special Diets Services, Witham, U.K.), ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12/12 h

Route of administration:

oral: feed

Vehicle:

other: Rat and Mouse SQC Expanded Diet No. 1 Ground

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The test material was incorporated into the diet at concentrations of 2250, 6000 and 15000 ppm as follows:
A known amount of test material was mixed with a small amount of basal laboratory diet for 19 min. at a constant speed setting in a Hobart QE200 mixer. This pre-mix was then added to a larger amount of basal laboraotry diet and mixed for further 30 min. at a constant speed setting in a Hobart M-802G mixer. The diet was staored in labelled, double black plastic bags in labelled, covered plastic bins not in use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material dietary admixture and were analysed for concentration and homogeneity of the test item at Safepharm Analytical Laboratory.
The test item concentration in the test dietary admixtures was determined by gas chromatography.
A. Homogeneity Determinations:
The dietary admixtures were sampled from the middle and two opposite sides in triplicate and anylysed.
B. Stability Determinations:
The dietary admixtures were sampled initially and then after storage at ambient temperature in the dark for six weeks.
C. Verification of Test Material Dietary Admixture Concentrations:
The test material dietary admixtures were sampled and analysed within 3 days of preparation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuously

Remarks:

Doses / Concentrations:
2250 ppm (227 mg/kg bw/d)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
6000 ppm (505 mg/kg bw/d)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
15000 ppm (1564 mg/kg bw/d)
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dietary concentrations were chosen based on the results of the range-finding palatability study.

The rat was elected for this study as it is a readiliy available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Positive control:

none

Observations and examinations performed and frequency:

- Clinical observations:
All animals were examined for overt signs of toxicity, ill-health or behavioural change once daily.

- Bodyweight:
Individual bodyweights were recorded on day 0 (the day before the start of treatment) and on days 7, 14, 21 and 28. Bodyweights were also recorded at necorpsy.

- Food consumption:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

- Water consumption:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

- Laboratory investigations:
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained from cardiac puncture prior to necopsy on day 29. Animals were not fasted prior to sampling.

a. Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin, Erythrocyte count, Haematocrit, Erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), total Leucocyte count, differential Leucocyte count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils), Platelet count.
Clotting time was assessed by "Hepato Quick" using samples collected into Sodium citrate solution (0.11 mol/L).

b. Blood cheimistry
The following parameters were measured on plasma from blood collected into tubes containing Lithium heparin anti-coagulant:
Urea, Glucose, total protein, Albumin, Albumin/Globuin ratio, Sodium, Potassium, Chloride, Calcium, inorganic Phosphorus, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatinine, total Bilirubin

Sacrifice and pathology:

Pathology
On completion of the dosing period all animals were killed by intravenous overdose of Sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external examination, and any macroscopic abnormalities were recorded.

A. Organ weights
The following organs, removed from animals that were killed at the end of the study were dissected free from fat and weighed before fixation:
Adrenals, Brain, Gonads, Heart, Kidneys, Liver, Pituitary, Spleen

B. Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% Formalin:
Adrenals, Aorta, Bone and bone marow, Brain, Caecum, Colon, Duodenum, Eyes, Muscle, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (cervical and mesenteric), Skin, Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder, Uterus.
The following preserved tissues from all control and high dose animals were prepared in parffin blocks, sectioned at nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination:
Adrenas, Hearts, Kidneys (all dose groups), Liver, Spleen and Testes.

Since there were indications of treatment-related renal changes, exmination was subsequently extended to include similarly prepared sections of kidney from all animals in the remaining dose groups.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analsis of variance incorporating "F-max" test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal-Wallis non-parametric analysis of variance and Mann Whitney U-Test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

corticomedullary mineralisation in kidneys (females)

Histopathological findings: neoplastic:

no effects observed

Details on results:

- Mortality
There were no deaths during the study.

- Clinical observations
There were no clinically observable signs of toxicity detected in test or control animals during the study.

-Body weight
All animals showed normal gains in body weight throughout the study period. Body weight gain in test animals were comparable with that seen in controls.

- Food consumption
No adverse effect on food consumption was detected. Food efficiency in test animals was comparable with that seen in controls.

- Water consumption
Visual inspection of water bottles revealed no overt intergroup differences.

- Haematology
No treatment-related changes were detected in the heamatological parameters measured.
Males from all treatment groups showed a slight but statistically significant reduction in mean corpuskular haemoglobin concentration. None of the individual values were outside the normally expected range for rats of the strain and age used, and the reduction was considered to be attributable to a slightly elevated control group mean rather than an effect of treatment.
A number of statistically significant changes were detected in males from various treatment groups, including the 15000 ppm treatment group, but showed no clear dose relationship and were, therefore, considered not to be toxicologically significant. These included an increase in mean corpuscular volume for males treated with 15000 or 2250 ppm, a reduction in neutrophil count for males treated with 6000 or 2250 ppm and an increase in lymphocyte count for males treated with 2250 ppm.

- Blood chemistry
No treatment-related changes were detected in the blood chemical parameters measured. Males treated with 15000 ppm showed a statisitcally significant increase in inorganic phosphorus when compared with controls. None of the individual values were outside the normally expected range for rats of the strain and age used and, in isolation, this increase was considered not to be toxicologically important.
The remaining statisitaclly significant intergroup differences, increases in total protein and plasma creatinine concentrations, were confined to males treated with 6000 and/or 2250 ppm. These differences were not dose-related and, as such, were considered to be of no toxicological sigificance.

- Pathology
A. Organ weights
There were no changes in the organ weights measured which could be considered attributable to treatment with the test material. Males treated with 15000 ppm or 2250 ppm showed a reduction in testes weight relative to bodyweight, when compared with controls. No clear dose relationship was demonstrated and this reduction was, therefore, considered not to be toxicologically significant.
The remaining statistically significant intergroup differences were confined to animals of either sex treated with 6000 ppm and involved a reduction in relative adrenal weight for males and an increase in relative kidney weight for females. In the absence of any dose-relationship, these differences were considered to be of no toxicological importance.

B. Necropsy
No macroscopic abnormalities were detected at terminal kill.

C. Histopathology
Treatment-related changes were observed. An increased incidence of focal corticomedullary mineralisation was observed for female rats dosed at 15000 ppm or 6000 ppm of the test material. Although corticomedullary mineralisation is a common spontaneous finding in the kidneys of laboratory maintained female rats, no control animals were affected in this study, and a relationship to treatment cannot be excluded. There were no associated blood chemical changes indicative of renal dysfunction and the macroscpic changes were considered not ot represent any adverse effect on the health of the animals.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Dose descriptor:

NOAEL

Effect level:

1 564 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: macroscopic change were not considered to represent any adverse effect

Critical effects observed:

not specified

Conclusions:

Dietary administration of Myristyl phosphate to rats for a period of 28 days at dietary concentrations of up to 15000 ppm (corresponding to an actual uptake of 1564 mg/kg bw/d) did not reveal any adverse health effects. Therefore, the NOAEL is considered to be 1564 mg/kg bw/d.

Executive summary:

A read across to the structural analogue, Phosphoric acid, tetradecyl ester (Myristyl phosphate), was performed. For read across justification please refer to the attached document "Phosphoric acid, hexadecyl ester".

Myristyl phosphate was administered by dietary admixture to three groups, each of five male and five female Sprague-Dawley CD rats, for 28 days at dietary concentrations of 2250, 6000 and 15000 ppm (equivalent to an estimated mean achieved dose level of 227, 505 and 1564 mg/kg bw/d respectively). A further group of five males and five females was given untreated laboratory diet to serve as control. Clinical signs, body weight, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no deaths during the study. No clinical observable signs of toxicity were detected in test or control animals throughout the study period. No adverse effect on body weight development was detected. No adverse effect on food and water consumption was detected. No treatment-related effects on haematology, blood chemistry and organ weights were reported. There were no macroscopic abnormalities. Females treated with 15000 or 6000 ppm showed an increased incidence of focal corticomedullary mineralisation in the kidneys. Although corticomedullary mineralisation is a common spontaneous finding in the kidneys of laboratory maintained female rats, no control animals were affected in this study, and a relationship to treatment cannot be excluded. This observation is considered to be a secondary effect due to the increased intake of phosphorus ester which is assumed to be hydrolysed to the corresponding alcoholic compound and phosphate. The elevated systemic phosphate level might lead to the generation of calcium phosphate which has the potential to precipitate observable a minimal to slight corticomedullary mineralisation. Since no associated blood chemical changes indicative of renal dysfunction were reported and macroscopic changes were considered not to represent any adverse effect on the health of the animals the NOAEL is set at 15000 ppm corresponding to 1564 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 564 mg/kg bw/day

Quality of whole database:

GLP conform guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

Relevant NOAEL (OECD 407, diet, rat): 1564 mg/kg bw/d

 

A read across to the structural analogue, Phosphoric acid, tetradecyl ester (Myristyl phosphate), was performed. For read across justification please refer to IUCLID Chapter 7.5.1.

Myristyl phosphate was administered by dietary admixture to three groups, each of five male and five female Sprague-Dawley CD rats, for 28 days at dietary concentrations of 2250, 6000 and 15000 ppm (equivalent to an estimated mean achieved dose level of 227, 505 and 1564 mg/kg bw/d respectively). A further group of five males and five females was given untreated laboratory diet to serve as control. Clinical signs, body weight, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no deaths during the study. No clinical observable signs of toxicity were detected in test or control animals throughout the study period. No adverse effect on body weight development was detected. No adverse effect on food and water consumption was detected. No treatment-related effects on haematology, blood chemistry and organ weights were reported. There were no macroscopic abnormalities. Females treated with 15000 or 6000 ppm showed an increased incidence of focal corticomedullary mineralisation in the kidneys. Although corticomedullary mineralisation is a common spontaneous finding in the kidneys of laboratory maintained female rats, no control animals were affected in this study, and a relationship to treatment cannot be excluded. This observation is considered to be a secondary effect due to the increased intake of phosphorus ester which is assumed to be hydrolysed to the corresponding alcoholic compound and phosphate. The elevated systemic phosphate level might lead to the generation of calcium phosphate which has the potential to precipitate observable a minimal to slight corticomedullary mineralisation. Since no associated blood chemical changes indicative of renal dysfunction were reported and macroscopic changes were considered not to represent any adverse effect on the health of the animals the NOAEL is set at 15000 ppm corresponding to 1564 mg/kg bw/d.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The OECD 407 study was selected as relevant available repeated dose toxicity study due to its reliability and since it provides a sensitive NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure/physical form.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure/physical form.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in two Guinea pig maximization tests
- due its non skin irritant and its physico-chemical properties (combination of its polar character (phosphoric molecule center) and the long extent of the alkyl chain) it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the intact skin barrier.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because
- no systemic effects or other evidence of absorption were observed in two Guinea pig maximization tests
- due its non skin irritant and its physico-chemical properties (combination of its polar character (phosphoric molecule center) and the long extent of the alkyl chain) it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the intact skin barrier.

Justification for classification or non-classification

Due to the NOAEL of 1564 mg/kg bw/day in an OECD 407 repeated dose toxicity study in rats performed with the structural analogue Phosphoric acid, tetradecyl ester, Phosphoric acid, hexadecyl ester does not have to be classified regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the Packaging Regulation (1272/2008/EC).