Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

Endpoint summary

Description of key information

- Oral route (rat, gavage, 80 days): NOAEL = 200 mg/kg bw/d (based on adrenal gland effects)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2008 - 11 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

except during study days -1 to 16, and except for the absence of chemical analyses of dosage forms

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder
- Age at first treatment: approx. 10 weeks
- Weight at first treatment (mean): M=408 g, F=269 g
- Housing: individually, except during pairing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Relative humidity: 50 ± 20%
- Light/dark cycle: 12h/12h
- Ventilation: 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: beginning: 22 April 2008 / end: up to 11 June 2008

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
test item was ground, heated to 80°C, mixed with vehicle heated to 80°C, forming a suspension.
The test item dosage forms were prepared daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): lipophilicity of the substance
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
- Lot/batch no. (if required): 057K6093
- Purity: not indicated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Absence of a practical method of analysis

Duration of treatment / exposure:

from 2 weeks before mating until the end of mating (males: total of 39 days) or day 5 pp (females: total of 43-51 days)

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (m/f)
Basis:
other: Nominal per gavage

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in a previous 14-day toxicity study in the rat (see 7.5.1) no relevant adverse effects occured at the dose-levels of 100, 300 or 1000 mg/kg/day.

- Rationale for animal assignment: computerized stratification procedure (average body weight of each group is similar)

- Satellite and post-exposure recovery period: not performed.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule: day 1 and then weekly; and in F also days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum

FOOD CONSUMPTION for each animal: Yes
- Time schedule: weekly (last 7-day consumption period)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5M+5F

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5M+5F

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: at the end of the treatment period for M, on day 5 post-partum for F
- Dose groups that were examined: all
- Battery of functions tested: behavior / reflexes / sensory activity / grip strength / motor activity / rectal temperature

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, adults and pups of all groups
ORGAN WEIGHTS: Yes, adults of all groups
HISTOPATHOLOGY: Yes, adults in control and high-dose

all : see Table 1

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related deaths or sacrifices (deaths were due to gavage errors, cranial hematoma or non-pregnancy).
Hypersalivation and reflux at dosing were observed in all groups and may be related to the vehicle and/or the test item but are non-adverse

BODY WEIGHT GAIN:
All female dose-groups gained less body weight than controls during the premating period, but this did not continue during gestation and lactation and was therefore considered to be non-adverse.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: NOAEL = highest tested dose. All female dose-groups gained less body weight than controls during the premating period, but this did not continue during gestation and lactation and was therefore considered to be non-adverse.

Critical effects observed:

not specified

Not necessary

Conclusions:

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The test item, LCE07103, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no treatment-related deaths during the study and the only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but are non-adverse. All female groups treated with LCE07103 gained less body weight during the premating period but this did not continue during gestation and lactation and was therefore considered to be non-adverse. There were no effects at any dose-level on food consumption. The Functional Observation Battery, motor activity assessment, hematology and blood biochemistry revealed no treatment-related effects. No treatment-related macroscopic or microscopic findings were noted and there were no treatment-related organ weight changes.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: preliminary study, only short summary available

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

No guideline followed: preliminary study

GLP compliance:

no

Remarks:

preliminary study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

olive oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (m/f)
Basis:
other: nominal per gavage

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Dose descriptor:

other: High-dose selected for definitive study

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: dose retained = highest tested dose, without relevant effects.

Critical effects observed:

not specified

No unscheduled deaths occurred during the study.

Hypersalivation was observed in the majority of males and females treated at all dose-levels, including the controls. This was observed mainly during week 2 but incidences were recorded during week 1. Reflux at dosing was observed in one control female and one female treated at 1000 mg/kg/day on one occasion only.

Mean body weight gains were variable due to the limited number of animals per group. Over the treatment period there were no effects on mean male body weight gain at any dose-level; the differences from the controls were 0%, +14% and -4% at 100, 300 and 1000 mg/kg/day respectively.

Females treated with LCE07103 showed minimally lower mean body weight gain during the first week of dosing and then at 300 and 1000 mg/kg/day moderately lower mean body weight gains during the second week of dosing. The differences compared with the controls over the treatment period were -18%, -50% and -35% at 100, 300 and 1000 mg/kg/day, respectively. There was high inter-animal variability and no clear dose-relationship. There were no effects of treatment with the test item on mean male food consumption; all groups had values comparable with the controls during both weeks of treatment. Female groups treated at 300 or 1000 mg/kg/day had slightly lower mean food consumption during the second week of treatment when compared with the controls, however there were no effects during the first week and there were no effects at 100 mg/kg/day during either week. There were no relevant macroscopic post-mortem findings.

Conclusions:

Under these experimental conditions, the high dose-level for the OECD 422 study was selected as 1000 mg/kg/day as this dose was considered high enough to elicit effects but not to result in mortality during the dosing period of up to 9 weeks.

Executive summary:

The test item, LCE07103, was administered once daily by gavage to rats at the dose-levels of 100, 300, and 1000 mg/kg/day for 14 days.

Effects at all dose-levels were limited to hypersalivation, which may be related to the vehicle and/or the test item but is considered as non-adverse, and lower mean female body weight gains and food consumption at 300 and 1000 mg/kg/day and, to a lesser extent at 100 mg/kg/day. Effects on body weight gain were observed during both weeks of treatment but were more noticable during the second week and food consumption was only affected during the second week of treatment and then only at 300 and 1000 mg/kg/day.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Well-conducted study according to a protocol very similar to OECD guideline 408, but with a treatment duration of 26 weeks

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: ~21-28 days at purchase
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: 5/cage in stainless steel cages, containing absorbent paper
- Diet (e.g. ad libitum): expanded rodent diet (Special Diets Services, UK), ad libitum
- Water (e.g. ad libitum): public supply (Suffolk Water Company, UK), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material heated to approx. 80 deg C
- vehicle heated to approx. 75 deg C
- vehicle added to test material while being magnetically stirred at high speed
- resulting 20% (w/w) suspension homogenized and slowly cooled to below 60 deg C
- when cooled to 30 deg C, suspension slowly homogenized again for >=2 min
- cooled to room temp.
- stored at 13 deg C
- prepared once weekly
- 20% suspension used for top dose; for low and mid doses, suspension magnetically stirred and aliquots taken for dilution on day of use; constant dose volume of 5 ml/kg bw per dose
- dilutions mixed by hand swirling followed by magnetic stirring

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 1%
- Amount of vehicle (if gavage): 5 ml/kg bw per dose
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: additional 10/sex per treatment group for toxicokinetic study, 6/sex controls
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): random

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS (including mortality): Yes
- Time schedule: >=twice daily
- Cage side observations included: evidence of reaction to treatment or moribund condition, evidence of ill health such as blood or loose faeces

DETAILED CLINICAL OBSERVATIONS: Yes, individual observations
- Time schedule: once daily during week 1, twice weekly during weeks 2 to 4, once weekly during weeks 5 to 13, once every 2 weeks from week 14 onwards

BODY WEIGHT: Yes
- Time schedule for examinations: pre-study, weekly during the study or more frequently if appropriate (for animals in moribund condition), at necropsy

FOOD CONSUMPTION:
- Food consumption for each cage determined: Yes

FOOD EFFICIENCY:
- Weekly group mean food conversion efficiencies calculated from the consumption and body weight gain data: Yes, for the first 14 weeks of treatment

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes, prestudy and at weeks 12 and 25

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 14 and 26
- Anaesthetic used for blood collection: Yes - halothane/nitrous oxide
- Animals fasted: Yes
- How many animals: 10/sex per dose level
- Parameters examined: packed cell volume, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, mean cell haemoglobin concentration, mean cell haemoglobin, mean cell volume; blood film samples examined for abnormal morphologhy and unusual cell types including normoblasts; prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 14 and 26
- Animals fasted: Yes
- How many animals: 10/sex per dose level
- Parameters examined: alkaline phosphatase activity, alanine amino transferase, aspartate amino transferase, gamma-glutamyl transpeptidase, glucose, total bilirubin, total cholesterol, urea, total triglyceride, total protein, electrolyte levels (Na, Cl, Ca), inorganic phosphorus, electrophorectic protein, creatine concentration

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 12 and 25
- Metabolism cages used for collection of urine: No data
- Animals fasted: No, but water deprived
- Parameters examined: pH, protein, glucose, ketones, bilirubin, urobilinogen, blood, specific gravity, appearance, volume

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- Bone marrow samples from femur: myeloid:erythroid ratio, cellularity and composition of marrow

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: adrenals, brain, kidneys, liver, lungs (with main stem bronchi), ovaries, pituitary, prostate, spleen, testes, thymus, thyroid (with parathyroids), uterus (with cervix)

HISTOPATHOLOGY: Yes - adrenals, brain, eyes, optic nerve, femur, heart, kidneys, liver, lungs, seminal vesicles, spinal cord, stomach, thyroid, uterus

Other examinations:

Satellite group for toxicokinetic study:
- Blood taken from satellite groups (3M+3F) non-fasted  on days 1, during weeks 13 and 26 at 0.5, 1, 2, 4, 8 and 24 hours after  dosing.

Statistics:

Bartlett's test for homogeneity of variance (organ weights, body weight changes); if significant, Behrens-Fisher test, otherwise Dunnett's test.
Two-tailed Fisher's exact test (macroscopic/microscopic pathological findings).
Student's t-test (haematology, clinical chemistry, urinalysis).

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- one male in the mid-dose group died at week 25 (examination suggested aspiration of test material through mis-dosing; not considered to be treatment-related)
- no other clinical signs of systemic toxicity or mortality

BODY WEIGHT AND WEIGHT GAIN
- no effects

FOOD CONSUMPTION
- presumably no effects

FOOD EFFICIENCY
- no effects

WATER CONSUMPTION
- not examined

OPHTHALMOSCOPIC EXAMINATION
- no effects

HAEMATOLOGY
- no effects
- no effects seen in bone marrow smears

CLINICAL CHEMISTRY
- no effects

URINALYSIS
- no effects

NEUROBEHAVIOUR
- not examined

ORGAN WEIGHTS
- no effects

GROSS PATHOLOGY
- no effects

HISTOPATHOLOGY: NON-NEOPLASTIC
- no effects

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- no effects

HISTORICAL CONTROL DATA (if applicable)
- no data

OTHER FINDINGS
- toxicokinetic study: concentrations of behenyl alcohol in the blood were measured on  day 1 and in weeks 13 and 26; maximum mean plasma concentration (Cmax) of behenyl alcohol occurred 1 hour after dosing in all males and most females; 24 hours after dosing plasma concentrations were below the limit of quantification  (<10ng/ml) at the 10 and 100 mg/kg dose levels while levels following  administration of 1000 mg/kg/day remained quantifiable on each sampling  day; Cmax in the top dose group ranged from 203.68 to 528.82 ng/ml throughout the duration of the study; statistically significant differences in area under the curve  (AUC24) were observed between males and females treated with 10 and 1000  mg/kg/day on day 1 and during week 13; the rate and extent of systemic  exposure to rats as shown by AUC24 and Cmax on day 1 and in weeks 13 and 26 increased with increasing dose level; increases were less than the proportionate dose increment and there was statistically significant evidence of non-proportionality on each sampling day.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Conclusions:

In a reliable study conducted according to a protocol very similar to OECD guideline 408, a repeated oral dose (26-week) NOAEL of 1000 mg/kg bw/day was determined in the rat. The study was performed in compliance with GLP. The result is a read across from 1-docosanol (CAS 661-19-8).

Executive summary:

Docosan-1-ol (CAS 661-19-8) was administered daily to groups of rats at levels up to 1000 mg/kg for 26 weeks. Body weight and food consumption was not affected by treatment. Haematology, clinical chemistry and gross necropsy investigations showed no evidence of toxicity. There were no treatment related microscopic changes.

Reference 5

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:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The general toxicity of C20-22 alkyl phosphate was evaluated in rats by oral route in one subacute toxicity study performed according to OECD 422 and in compliance with GLP. This study was scored as validity 1 according to Klimisch criteria and therefore was selected as the Key study. The results of this study were supported and completed by those observed in a subchronic toxicity study with Potassium salt of alkyl (C9-15) phosphate performed according to OECD 408 and in compliance with GLP (performed by oral route in the rats).

No data are available by dermal route or by inhalation.

OECD TG 422 with C20-22 alkyl phosphate:

C20 -22 alkyl phosphate was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

There were no treatment-related deaths during the study and the only clinical signs observed were hypersalivation and reflux at dosing, which were considered to be related to treatment with the test item but are non-adverse. All female groups treated with C20-22 alky phosphate gained less body weight during the premating period but this did not continue during gestation and lactation and was therefore considered to be non-adverse. There were no effects at any dose-level on food consumption. The Functional Observation Battery, motor activity assessment, hematology and blood biochemistry revealed no treatment-related effects. No treatment-related macroscopic or microscopic findings were noted and there were no treatment-related organ weight changes.

The NOAEL based on the absence of effects was 1000 mg/kg bw/d.

OECD 408 assay with Potassium salt of alkyl (C9-15) phosphate:

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 NOAEL based on adrenal gland effects was 200 mg/kg bw/d.

Justification for classification or non-classification

Based on the classification criteria of UN/EU GHS, and given the absence of signs of toxicity up to 200 mg/kg bw/d administered in rats by gavage during 90 days, no classification for repeat-dose toxicity is warranted.