Phosphoric acid, mono- and di-C16-18-alkyl esters

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-27 to 2009-03-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts. 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 a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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
- Age at study initiation: 5 weeks
- Weight at study initiation: mean 167 g for males (154 - 184 g) and mean 148 g for females (134 - 159 g)
- Fasting period before study: no
- Housing: individually in a stainless steel wire mesh cage
- Diet: Solid diet LaboMRstock of Nihon Nosankogyo Co., ad libitum
- Water: tap water filtered through a cartridge of 1 μm pore size and irradiated with UV light, ad libitum
- Acclimation period: males 7 days, females 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 to 24.5°C
- Humidity (%): 46 to 61%
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2008-11-27 To: 2009-03-06 (main groups), 2009-03-20 (recovery groups)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water (JP, Lot No. 181639, Kyoei Pharmaceutical Co.)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE: water
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 181639
- Purity:
The compound was administered using an injection syringe fitted with a Teflon stomach tube at a dose volume of 5 mL/kg once daily for 13 weeks (91 days). The administration was conducted in the morning and the control group received purified water (JP). The dosing volume was adjusted on the basis of the newest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Appendix 2 not in translation

Duration of treatment / exposure:

dosing period of 91 days, 14-day recovery period

Frequency of treatment:

once daily (in the morning)

Remarks:

Doses / Concentrations:
8 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
40 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Each group consisted of 10 males and 10 females. Further, 5 males and 5 females were added to the control group and 1000 mg/kg group as the satellite group to examine the reversibility of the change.

Control animals:

yes, concurrent vehicle

other: high dose recovery group

Details on study design:

- Dose selection rationale: dose-finding study, 30, 100, 300 and 1000 mg/kg each were administered to 4 male and 4 female rats for 14 days
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: examine the reversibility of changes
- Post-exposure recovery period in satellite groups: 14-days
- Section schedule rationale (if not random): random

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 4 times daily during the dosing period, namely before administration, immediately after administration, about 30 minutes to 1 hour after administration, and 4 hours after administration, and at least once daily during the recovery period.
- Cage side observations checked: lethality, external appearance and behavior

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
- Parameters checked in table No.1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 (immediately before dosing), Day 7, and at an interval of 7 days thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): once every week for 24-hour consumption
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time -weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During Week 13 (90th day of administration for males and 89th day of administration for females) for animals scheduled to be sacrificed at the end of the dosing period, and during recovery Week 2 (13th day of recovery for males and 12th day of recovery for females) for recovery animals
- Dose groups that were examined: 1000 mg/kg group and control group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day following the final dosing for surviving animals and on the day following the end of the recovery period for recovery animals
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes
- How many animals: 10 (main groups), 5 (recovery groups)
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day following the final dosing for surviving animals and on the day following the end of the recovery period for recovery animals
- Animals fasted: Yes
- How many animals: 10 (main groups), 5 (recovery groups)
- Parameters checked in table No.3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At Week 13 (85th to 86th day in both sexes) and Week 2 of the recovery period (8th to 9th day for males and 7th to 8th day for females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The animals to be sacrificed at the end of the dosing period were subjected to the following sensory response tests at Week 13 (90 days after starting dosing). Recovery animals were tested during Week 2 (9th day of recovery)
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity
- Parameters checked in table No.2 were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Statistics:

Statistical treatment Mean values and incidences were subjected to statistical treatment by the following method. Difference from the control group was considered significant when p is < 5%.
(1) Parametric data For multi-group comparison, Bartlett variance analysis was performed. When the variance was uniform, one-way analysis of variance was employed. When significant difference was noted, each group was compared with the control group by Dunnett test. When the variance was not uniform, analysis of nonparametric data was followed. For two-group comparison, F test was applied and when the variance was uniform, Student t test was used and when the variance was not uniform, Aspin-Welch t test was employed (body weight, body weight gain, food intake, grip strength, locomoror cavity, urine volume, urine specific gravity, hematology data, blood biochemistry data, organ weight).
(2) Nonparametric data For multi-group comparison, Kruskal-Wallis rank test was performed. When significant difference was noted, Dunnett test was used to compare each group with the control group. For two-group comparison, Mann-Whitney U test was employed (qualitative data in urinalysis, leucocyte percentage, graded incidence in histopathology).
(3) Categorical data Fisher’s exact probability test was employed (incidence of abnormal event in observation of general status, detailed clinical observation, sensory response test, ophthalmology, necropsy and histopathology). For the findings where graded score was used in histopathology, Mann-Whitney U test was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No death was observed.
200 mg/kg bw/d
- transient salivation in 3 males (Day 73) and 1 female (Day 71 and 72)
- abnormal respiratory sound in males

1000 mg/kg bw/d:
- transient salivation was in 12 males and 14 females (mostly after Day 50)
- abnormal respiratory sound in both sexes

BODY WEIGHT AND WEIGHT GAIN
1000 mg/kg bw/d
- body weight of males was consistently lower than that of the control group
- body weight gain during the dosing period was significantly less

recovery group
- body weight of males was generally in parallel with that of the control animals.
- in females, mean body weight of the recovery group was lower than that of the control group; no difference in body weight gain

FOOD CONSUMPTION
- no statistically significant changes

OPHTHALMOSCOPIC EXAMINATION
- no abnormalities was observed

HAEMATOLOGY
- no statistically significant changes

CLINICAL CHEMISTRY
40 mg/kg bw/d
- A/G ratio significantly high in females

200 mg/kg bw/d
- A/G ratio was significantly low in males
- A/G ratio significantly high in females
- total protein was significantly high in males but no dose dependency was evident

1000 mg/kg bw/d
- urea nitrogen was significantly low in males
- A/G ratio was significantly low in males
- A/G ratio significantly high in females

recovery group
- no change in urea nitrogen or A/G ratios

URINALYSIS
1000 mg/kg bw/d
-statistically significant lower pH in both sexes
- Protein in females was significantly higher; as this was not accompanied by organic change, it was not considered to represent a toxicological effect
No change was observed in recovery animals.

NEUROBEHAVIOUR
- no treatment related effects

ORGAN WEIGHTS
1000 mg/kg bw/d
- absolute weight of the heart was significantly low,
- relative weight of the thyroid was significantly high in males
- relative weight of heart was significantly low
- absolute weight of the liver and thyroid was significantly high in females
These changes noted in the animals sacrificed at the end of the administration period were not observed in recovery animals. In the recovery group, absolute weight of the testis and epididymis was low in males and relative weight of the brain was significantly high in females.

GROSS PATHOLOGY
1000 mg/kg bw/d
- mild to moderate thickening of the forestomach mucosa in 9/10 animals in both sexes
- mild to moderate dilatation of cecum due to gas retention in 2 males.
- no abnormality was observed in the recovery animals of either sex

HISTOPATHOLOGY: NON-NEOPLASTIC
In the forestomach, mild to marked hyperplasia of squamous epithelium (diffuse thickening accompanied with advanced cornification of squamous epithelium) was noted in 8 males and 10 females at 1000 mg/kg bw/d, the incidences being statistically significant. Erosion was also observed in 5 males and 4 females.
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.
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. In the heart, thyroid, liver, testis, epididymis and brain which showed some change in organ weight measurement, no compound-related finding was obtained. In the animals sacrificed at the end of the recovery period, no change was recognized in the forestomach and only mild change was noted in the adrenal gland in 2 males and 2 females out of 5 each, and recovery or its tendency was evident.

Dose descriptor:

BMDL10

Effect level:

240.3 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: adrenal gland effects

Critical effects observed:

not specified

General status and lethality

Abnormal respiratory sound was noted in 5 males (after Day 42, sporadically) and in 1 female (Day 18 of dosing) at 200 mg/kg bw/d, in 15 males and in 14 out of 15 females at 1000 mg/kg bw/d (after Day 1 for both sexes). This sound was detected immediately after dosing and disappeared after 1 hour in some cases and in some cases by the next morning. In some cases, it appeared 1 hour after dosing and not detected the next morning but persisted for several days in some cases. Salivation was observed in 3 males (Day 73) and 1 female (Day 71 and 72) at 200 mg/kg bw/d and in 12 males and 14 females (mostly after Day 50) at 1000 mg/kg bw/d. This salivation was a transient phenomenon observed immediately after dosing resulting in mild moistening of the area around the mouth. Incidences of abnormal respiratory sound in males at 200 mg/kg bw/d and in both sexes at 1000 mg/kg bw/d were significantly different from that of the control group. No death was observed. Abnormal respiratory sound and salivation were not observed during the recovery period.

Detailed clinical observation

No statistically significant change was noted during the dosing period or recovery period.

Functional tests

1)     Sensory response tests

No statistically significant change was noted during the dosing period or recovery period.

 

2)     Grip strength and locomotor activity

No statistically significant change was noted in grip strength of forelimb/hind limb and locomotor activity during the dosing period or recovery period.

Body weight

At 1000 mg/kg bw/d, body weight of males was consistently lower than that of the control group throughout the dosing period and statistical significance was reached after Day 7 of dosing except Day 63 and Day 70. Body weight gain during the dosing period was significantly less. No statistically significant difference from the control group was noted in females at 1000 mg/kg bw/d. During the recovery period, body weight of males was generally in parallel with that of the control animals. No statistically significant change was noted in body weight at each measuring point and in body weight gain during the recovery period.. In females, mean body weight of the recovery group was lower than that of the control group and statistical difference was noted on Day 7 and Day 14 of recovery. There was no difference in body weight gain during the recovery period. 

Food intake

No statistically significant change was noted in the dosing groups during the dosing period or during the recovery period.

Ophthalmology

No abnormality was observed.

Urinalysis

At 1000 mg/kg bw/d, statistically significant lower pH was noted in both sexes. Protein in females was significantly higher. Change in protein in females at 100 mg/kg bw/d (2+ in 8) was within the variation of the control animals (-to 2+). No change was observed in recovery animals. 

Hematology

No statistically significant change was noted in the dosing groups at the end of the dosing period or at the end of the recovery period.

Blood biochemistry

At 1000 mg/kg bw/d, urea nitrogen was significantly low in males. A / G ratio was significantly low in males at 200 mg/kg bw/d or higher, significantly high in females at 40 mg/kg bw/d or higher. The change was not consistent in males or females and no clear dose dependency was noted. A / G ratio of individual animals was within the historical range of this research laboratory (males: n = 70, mean 0.91, 0.76 - 1.07; females: n = 70, mean 1.23, 0.87 - 1.58). No change was noted in albumin values. At 200 mg/kg bw/d, total protein was significantly high in males but no dose dependency was evident. Examination at the end of the recovery period showed no change in urea nitrogen or A / G ratios. ALP and sodium were significantly high in females but this change was not noted at the end of the dosing period. On examination of individual values, 2 ALP values (351 and 290mEq/L) were above the historical control range (ALP: females n = 70, mean 165, 77 - 254 IU / L; sodium females n = 70, mean 145, 141 - 150 mEq/L) but other values were within the historical control range. For ALP, the values of the control group happened to be low and the finding was considered incidental. 

Necropsy

At 1000 mg/kg bw/d, mild to moderate thickening of the forestomach mucosa was noted in 9 out of 10 animals in both sexes and mild to moderate dilatation of cecum due to gas retention was noted in 2 males. Statistical significance was noted in incidence of forestomach mucosal change in males and females in comparison with the control group. No abnormality was observed in the recovery animals of either sex.

Organ weight

At 1000 mg/kg bw/d, absolute weight of the heart was significantly low, relative weight of the thyroid was significantly high in males and relative weight of heart was significantly low and absolute weight of the liver and thyroid was significantly high in females. These changes noted in theanimals sacrificed at the end of the recovery periodwere not observed in recovery animals. In the recovery group, absolute weight of the testis and epididymis was low in males and relative weight of the brain was significantly high in females.

Histopathology

Compound-related effects were seen in the forestomach and adrenal gland. In the forestomach, mild to marked hyperplasia of squamous epithelium was noted in 8 males and 10 females at 1000 mg/kg bw/d, the incidences being statistically significant. This finding is characterised by diffuse thickening accompanied with advanced cornification of squamous epithelium. Erosion was also observed in 5 males and 4 females. There was no significant difference in the incidence of hyperplasia of squamous epithelium and erosion between both sexes. 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. In the heart, thyroid, liver, testis, epididymis and brain which showed some change in organ weight measurement, no compound-related finding was obtained.In the animals sacrificed at the end of the recovery period, no change was recognized in the forestomach and only mild change was noted in the adrenal gland in 2 males and 2 females out of 5 each, and recovery or its tendency was evident.

In addition to the above, the findings observed either in the control group alone or in both the control group and dosed groups, included the following: Foam cell accumulation in the lungs (both sexes), mineral deposition in the arterial wall (both sexes), small granuloma (males), hemorrhage (males), osseous metaplasia (males), cardiac muscle degeneration/fibrosis (males), hepatic minute granuloma (both sexes), fundic swelling of glandular stomach (both sexes), renal basophilic tubule (both sexes), cellular cast (males), hyaline cast (females), cortical lymphocyte infiltration (both sexes), epithelial eosinophilic body of proximal tubule (males), mineral deposition in the tubule (males), epithelial fibrous focus (females), vacuolar degeneration of adrenal zona fasciculata (both sexes), Rathke’s pouch remnant of the pituitary (females), ultimobranchial rest of the thyroid (both sexes), hemorrhage of the thymus (both sexes), extramedullary hematopoiesis (both sexes), and deposition of brown pigment (both sexes) of the spleen, and interstitial lymphocyte infiltration of the prostate gland (males). The incidence of these findings was not statistically significant. At 1000 mg/kg bw/d, atrophy of the pancreatic exocrine cells (females) and renal cortical fibrosis (males), isolated cyst (females), and mineral deposition at cortico-medullary junction (females) were recognized but these were seen only in 1, 1, 1 and 2 animals, respectively, and are known as spontaneously occurring lesions in rats. Therefore, they were considered incidental.

 

Conclusions:

A 13-week repeated oral dose toxicity study of Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts was conducted in Sprague-Dawley rats. Doses were 0 (control), 8, 40, 200 and 1000 mg/kg bw/day. Salivation and abnormal respiratory sound immediately after dosing were noted sporadically at 200 mg/kg bw/d in both sexes, and in relatively high incidence at 1000 mg/kg bw/d in both sexes. Body weight was low in males at 1000 mg/kg bw/d. No change was observed in food intake. On urinalysis, pH was low in both sexes at 1000 mg/kg bw/d. At the time of necropsy, thickening of the forestomach mucosa was noted in both sexes and dilatation of cecum due to gas retention was observed in males at 1000 mg/kg bw/d. On histopathological examination, hyperplasia and erosion of squamous cells of the forestomach were observed in both sexes at 1000 mg/kg bw/d and hypertrophy of the cortical glomerular zone in the adrenal gland was noted in both sexes at 200 mg/kg bw/d or higher doses. No adverse reaction was observed in detailed clinical observation, sensory response test, grip strength test, locomotor activity, ophthalmology, hematology, blood biochemistry or organ weight measurement. 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 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. The BMDL10 based on adrenal gland effcts was 240.3 mg/kg bw/d (BMD10 = 374.61 mg/kg bw/d).

Executive summary:

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.

 

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. 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. Thus, the forestomach effects are considered to be not substance-related and therefore not taken into account for the derivation of the dose descriptor.

 

Differing from the study authors, the BMDL10 was derived from this study. This approach was considered more adequate as it better reflects the dose-response curve as proposed in ECHA’s “Guidance on information requirements and chemical safety assessment Chapter R.8: Characterisation of dose [concentration]-response for human health.“ (version 2, December 2010).

 

The BMDL10 based on adrenal gland effects was 240.3 mg/kg bw/d (BMD10 = 374.61 mg/kg bw/d).

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rat.

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 - 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.

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 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 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, mono- and di- C16-18 (even numbered) alkyl esters 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.