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

Neurotoxicity

Description of key information

Subchronic (90-day) study oral (gavage), rat (Sprague-Dawley) m/f (OECD guideline 408, GLP): NOAELneurotoxicity: 1000 mg/kg bw/day (both sexes); read-across substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic 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:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents), including functional battery for neurobehaviour

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 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

Observations and clinical 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:

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: at Week 13 (90 days after starting dosing), recovery animals were tested during Week 2 (9th day of recovery)
Auditory response (startle response when the cages were mildly touched with tweezers), visual response (response when a rod was positioned closely to the face), touch response (response when lumbar is touched), pain response (response such as escaping or vocalization when tail is pricked with tweezers), pupillary response (response of pupil to light), and righting reflex (response when animals placed in supine position return to normal position).

LOCOMOTOR ACTIVITY: Yes
Grip strength of forelimb and hindlimb was measured during Week 12 (84th day of administration for both sexes) for animals scheduled to be sacrificed at the end of the dosing period, and during recovery Week 2 (13th day of recovery) for recovery animals (grip strength measuring apparatus for rats and mice, MK-380R/FR, Muromachi Kikai Co.). Locomotor activity was measured at the same period (locomotor activity measuring apparatus SUPERMEX, Muromachi Kikai Co.; far infrared ray emitted by animals is detected by a sensor and number of quarter-crossing in the apparatus during 60 minutes is counted).

Sacrifice and (histo)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

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

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:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: neurobehaviour

Remarks on result:

other:

Dose descriptor:

BMDL10

Effect level:

240.3 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects (adrenal gland)

Remarks on result:

other:

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 290 mEq/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 potassium salt of alkyl (C=9-15) phosphate 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 in both sexes, and in relatively high incidence at 1000 mg/kg in both sexes. Body weight was low in males at 1000 mg/kg. No change was observed in food intake. On urinalysis, pH was low in both sexes at 1000 mg/kg. 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. On histopathological examination, hyperplasia and erosion of squamous cells of the forestomach were observed in both sexes at 1000 mg/kg and hypertrophy of the cortical glomerular zone in the adrenal gland was noted in both sexes at 200 mg/kg or higher doses.

No adverse reaction was observed in hematology, blood biochemistry or organ weight measurement.

Concerning neurological toxicology and behavior toxicology, no toxic effect was seen in detailed clinical observation, sensory response test, grip strength test, or histopathological examination of neurological organs and tissues. No change was noted in ophthalmological examination.

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 potassium salt of alkyl (C=9-15) phosphate appears mainly in the adrenal gland and forestomach.

Executive summary:

In a subchronic toxicity study according to OECD guideline 408 Potassium salt of alkyl (C=9- 15) phosphate (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 hematology, blood biochemistry or organ weight measurement. Concerning neurological toxicology and behavior toxicology, no toxic effect was seen in detailed clinical observation, sensory response test, grip strength test, or histopathological examination of neurological organs and tissues. No change was noted in ophthalmological examination.  

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 potassium salt of alkyl (C=9- 15) phosphate appears mainly in the adrenal gland and forestomach.

 

The NOAEL for neurobehavioural effects is 1000 mg/kg bw/d in both sexes.

The BMDL10 for overall effects (adrenal gland, for details see repeated dose toxicity part of this study) is 240.30 mg/kg bw/d (corresponding BMD =374.61 mg/kg bw/d).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available study is a high quality guideline study 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 a read-across substance.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable data on neurotoxicity of PAE are available from a 90-day gavage study in rats on the read-across substance Phosphoric acid, C9-15 branched and linear alkyl esters, potassium salts.

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 and placenta. In human and most other mammals three isoenzymes of Alkaline phosphatase exist: intestinal ALP, placental ALP, tissue non-specific ALP (present in bone, liver, kidney, skin).

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

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

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

 

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 hematology, blood biochemistry or organ weight measurement. Concerning neurological toxicology and behavior toxicology, no toxic effect was seen in detailed clinical observation, sensory response test, grip strength test, or histopathological examination of neurological organs and tissues. No change was noted in ophthalmological examination.  

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 NOAEL for neurobehavioural effects is 1000 mg/kg bw/d in both sexes.

The BMDL10 for overall effects (adrenal gland, for details see repeated dose toxicity part of this study) is 240.30mg/kg bw/d (corresponding BMD =374.61 mg/kg bw/d).

There is no data gap in neurotoxicity. There is no reason to believe that results of the study would not be relevant for neurotoxicity in humans and, therefore, for risk assessment.

Justification for selection of effect on neurotoxicity via oral route endpoint:
OECD guideline study, no deviations, GLP; 90 d study

Justification for classification or non-classification

In conclusion, the results of the available data on neurotoxicity indicate that Phosphoric acid, mono- and di- C11-14 (linear and branched) alkyl esters does not need to be classified according to Directive 67/548/EEC as well as CLP, EU GHS (Regulation 1272/2008/EC) and therefore labelling is not necessary.