Phosphoric acid C12-alkyl and C18-alkyl esters, potassium salts

Endpoint summary

Description of key information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening with the structural analogue Phosphoric acid, dodecyl ester, sodium salt the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity was considered to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: absence of local (irritation) effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of local (irritation) effects

Key result

Critical effects observed:

not specified

Conclusions:

Based on the results of the available study together the NOAEL of the test item was found to be 1000 mg/kg bw/d.

Executive summary:

In this study conducted according to OECD Guideline 422 the test item was administered at 0 (control group), 250, 500, or 1000 mg/kg bw/d (vehicle: olive oil) to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total), and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw/d groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, results of functional tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Histopathological examinations at the end of the administration period showed, upon gross observation, indentation of the anterior stomach in the 250 mg/kg bw/d and higher dose groups, and rough mucosa or white foci in the anterior stomach in the 500 mg/kg bw/d and higher dose groups, and upon histological examination, erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema of the submucosal tissue in the 250 mg/kg bw/d and higher dose groups. On the basis of these results, the no observed effect level of the test item was judged to be less than 250 mg/kg bw/d in relation to repeated-dose toxicity in both males and females. The observed effects are limited to the forestomach and stomach of the test animals. They are not considered adverse in regard to systemic toxicity, as they are a result of the local irritation of the test item after direct contact to the mucosa due to the gavage application, during which massive amounts of the test substance are applied at once. A supplementary study was conducted to assess the no observed effect levels, which revealed a NOEL for male animals of 125 mg/kg bw/d and a NOEL for female animals of 62.5 mg/kg bw/d.

As no adverse systemic effects were observed, a NOAEL of 1000 mg/kg bw/d was determined.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: supplied by the Office of Chemical Safety, Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (observed temperature, 15°C-28°C) under hermetically sealed conditions
- Stability under test conditions: It was confirmed at Bozo Research Center that 50 and 200 mg/mL suspensions of the test substance (vehicle: olive oil) remained stable for 24 hours at room temperature after storage in light-protected containers in a cold dark place (refrigerator) for 7 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: An appropriate amount of the test substance for each concentration was weighed and suspended in olive oil (Maruishi Pharmaceutical Co., Ltd., batch No. 4604, 4720) in an agate mortar to prescribed concentrations (50, 100, and 200 mg/mL). The test suspensions were prepared at a frequency of at least once every 7 days and stored in a cold dark place (refrigerator, observed temperature: 3°C-5°C) in light-protected containers (brown glass bottles) until use.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi Breeding Center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 wks
- Weight at study initiation: 338 to 395 g (males) and 219 to 256 g (females)
- Housing: Animals were housed individually in a bracket type metal wire cage (250 mm x 350 mm x 200 mm). During the mating period, one pair of male and female animals was housed per cage. From gestation day 17 to day 5 of lactation, the animals were housed individually in a plastic Econ cage (340 mm x 400 mm x 185 mm) with bedding (White Flakes: Charles River Japan).
- Diet: The animals were allowed free access to Solid chow NMF (nonsterilized: Oriental Yeast Co., Ltd., batch numbers 040713, 040806, 040913)
- Water: tap water ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 37-77
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the test substance for each concentration was weighed and suspended in olive oil (Maruishi Pharmaceutical Co., Ltd., batch No. 4604, 4720) in an agate mortar to prescribed concentrations (50, 100, and 200 mg/mL).
- Rate of preparation of diet (frequency): at least once every 7 days

VEHICLE
- Lot/batch no.: 4604, 4720

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suspensions of each concentration used for administration at week 1 and on the last week of administration were analyzed by HPLC at the Bozo Research Center. The results showed that for all the suspensions tested, the percentage of the test substance relative to the nominal value was in the range of 96.5% to 105.0%, with a C.V. in the range of 1.0% to 5.3%, which were within the acceptable range (concentration, nominal value ± 10%; C.V., < 10%).

Analytical method:
The test sample (dosing suspension), 1 mL, was diluted with 60 vol% of THF solution to 10 mL and centrifuged (2000 rpm, 1000 x g, 20°C, 5 minutes); then, 1 mL of the lower layer was diluted to 5 mL with the mobile phase of HPLC. The diluted solution was filtered through a Milex HV filter and the filtrate was subjected to measurement by the HPLC system. Single test samples were taken from the upper, middle and lower layers of the dosing suspension.

HPLC system:
Pumping unit: LC-10AT (Shimadzu Corp.)
Online degasser: DGU-14A (Shimadzu Corp.)
Autoinjector: SIL-10AXL (Shimadzu Corp.)
Colum oven: CTO-10A (Shimadzu Corp.)
Detector: RID-10A (Shimadzu Corp.)
System controller: SCL-10A (Shimadzu Corp.)
Data processor: Millenium32 Chromatography Manager
(Nihon Waters K.K.)

HPLC conditions:
Column: CAPCELL PAK C18 UG120
(4.6 mm I.D. x 250 mm, 5 ^m. Shiseido Co., Ltd.)
Column temperature: 50°C (set temperature of the column bath)
Mobile phase: 0.25 mol/L sodium perchlorate solution (in methanol/purified water mixture [85 : 15,v/v], pH 2.5)
Flow rate: 1.0 mL/min
Detection: RI
Injection volume: 10 µL
Test sample temperature: Room temperature

Duration of treatment / exposure:

The duration of administration was 14 days before mating, 14 days during the mating period, and 14 days after the mating period, that is, 42 days in total, for the males of the main group and the males and females of the recovery group, and 14 days before mating and up to day 4 of lactation throughout the mating and gestation periods, that is, 42 to 45 days in total, for the females of the main group.
The recovery period for the males and females of the recovery group was 14 days after the end of administration, during which period the test substance was not given to the animals.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previous study 14-day repeated-dose oral toxicity study ofthe test item in rats (a preliminary study) (doses: 125, 250, 500, and 1000 mg/kg bw/d), administration of the test substance did not produce any effect even at 1000 mg/kg bw/d, the level defined as the limiting dose by the OECD Guideline for Testing of Chemicals 422. Therefore, 1000 mg/kg bw/d was set as the highest dose, and doses of 500 and 250 mg/kg bw/d were derived by dividing by a common factor of 2. A total of 3 doses were thus set up.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times a day
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the start of administration for all animals, once every week during the administration period for the males of the main group, once every week during the pre-mating administration and mating periods, and on predetermined days (gestation days 1, 7, 14 and 20, day 4 of lactation) during the gestation period and the lactation period for the females of the main group, and once every week during the administration and recovery periods for the animals of the recovery group. Function tests, measurement of the grip strength, and measurement of the spontaneous motor activity were performed on the last week of administration (day 39 of administration) for the males of the main group, after F1 necropsy on day 4 of lactation (day 42-45 of administration) for the females of the main group, and on the last week of administration (day 39 of administration) and last week of the recovery period (day 11 of recovery) for the males and females of the recovery group.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, and on the day of necropsy for the males of the main group, on days 1, 4, 8, 11 and 14 of the recovery period and on the day of necropsy, in addition to the days of measurement for the males of the main group, for the males and females of the recovery group, and on days 1, 4, 8, 11 and 15 of administration (and days 18, 22 and 25 of the administration period as well as in non-copulated animals), days 0, 4, 7, 11, 14, 17 and 20 of gestation, and days 0 and 4 of lactation for the females of the main group

FOOD CONSUMPTION AND COMPOUND INTAKE
The amount of food remaining relative to that supplied on the previous day was measured on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration for the males of the main group, on days 1, 4, 8, 11 and 14, in addition to the days of measurements for the males of the main group, for the males and females of the recovery group, on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation, and days 2 and 4 of lactation for the females of the main group. Food consumption per animal was calculated from the data thus obtained.

Sacrifice and pathology:

SACRIFICE
All of the 5 male and female animals in each group from which blood samples were collected for the hematology and blood chemistry tests on the day after the last day of administration and on the last day of the recovery period were exsanguinated to death after the blood collection, and all the other animals were exsanguinated to death by dissecting the abdominal aorta under ether anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Males of the 1000 mg/kg bw/d group in the main group showed a significant increase of the defecation frequency during weeks 1 and 2 of administration, which was a very mild transient change and considered to be within normal range.
No abnormalities were observed in the other parameters in any of the animals in either the main group or the recovery group. No significant differences were observed in the standing frequency between the control group and any of the treatment groups.
In the recovery group, one male in the 1000 mg/kg bw/d group showed decreased spontaneous motor activity from day 37 of administration up to day 7 of the recovery period, and wheezing from day 37 of administration until the end of the recovery period, these symptoms were judged to be due to inadvertent administration of the test substance into the upper respiratory tract.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly greater increase in body weight was observed in the females of the 250 and 1000 mg/kg bw/d groups during the lactation period, but the increase was not dose-related. In the recovery group, males in the 1000 mg/kg bw/d group showed decreased body weight gain during the administration period and decreased body weight (-21 g) during the recovery period. This was caused by the abnormality in 1 out of the 5 animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Administration of the test substance did not have any effect on the food consumption in the males or females of either the main group or the recovery group. A significant increase was observed on days 2 and 4 of lactation in the females of the 250 mg/kg bw/d dose group in the main group, but this was not dose-related.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period, a significant increase in the serum level of fibrinogen was observed in the females of the 250 mg/kg bw/d group and a significant decrease in the percentage of lymphocytes and significant increase in the percentage of segmented neutrophils were observed in the females of the 500 mg/kg bw/d group. However, none of these changes were observed in the 1000 mg/kg bw/d group, suggesting that they were within the range of physiological variations.
At the end of the recovery period, a significant increase in the mean corpuscular volume of the red blood cells, significant decrease of the platelet count, significant increase in the percentage of lymphocytes, and significant decrease in the percentage of segmented neutrophils were observed in females of the 1000 mg/kg bw/d group. However, these changes were not observed at the end of the administration period, which suggested that they were within the range of physiological variations.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period, a significant increase in the serum level of ALT was observed in the males of the 1000 mg/kg bw/d group. A significant decrease of the serum level of inorganic phosphorus was observed in the males of the 250 mg/kg bw/d group. However, since the decrease was not dose-related, it was considered to be within the range of physiological variations.
At the end of recovery period, a significant increase in the serum level of total protein was observed in the females of the 1000 mg/kg bw/d group. However, since no such change was observed at the end of the administration period, the increase was considered to be within the range of physiological variations.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormalities were observed in the qualitative parameter values in any of animals in either the main group or in the recovery group.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No dose-related changes in either direction (increase or decrease) were observed in either the absolute or the relative weight. Although significant differences in the weights of some organs were observed, they were considered to be within the range of normal variations, because they were neither dose-related nor were observed at the end of the administration period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross observation showed indentation of the anterior stomach in the animals of the 250 mg/kg bw/d and higher dose groups, and rough mucosa and/or white foci in the anterior stomach in the animals of the 500 mg/kg bw/d and higher dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological observation showed erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema in the submucosal tissue, suggesting the irritability of the test substance in the animals of the 250 mg/kg bw/d and higher dose groups,

Histopathological findings: neoplastic:

no effects observed

Details on results:

On the basis of these results, the no observed effect level was judged to be less than 250 mg/kg bw/day for repeated-dose toxicity in both males and females.
In order to determine the no observed effect level in repeated-dose toxicity, a supplementary study was conducted in which the test substance, 62.5 and 125 mg/kg bw/d, was administered to male and female rats of the same strain for 45 days. As a result, edema was observed in the submucosal tissue of the anterior stomach in females of the 125 mg/kg bw/day group.1 On the basis of the main study and the supplementary study, the no observed effect level in repeated-dose toxicity was judged to be 125 mg/kg bw/day in males and 62.5 mg/kg bw/day in females.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: absence of local (irritation) effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of local (irritation) effects

Key result

Critical effects observed:

not specified

Conclusions:

Based on the results of the available study together the NOAEL of the test item was found to be 1000 mg/kg bw/d.

Executive summary:

In this study conducted according to OECD Guideline 422 the test item was administered at 0 (control group), 250, 500, or 1000 mg/kg bw/d (vehicle: olive oil) to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total), and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg bw/d groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, results of functional tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Histopathological examinations at the end of the administration period showed, upon gross observation, indentation of the anterior stomach in the 250 mg/kg bw/d and higher dose groups, and rough mucosa or white foci in the anterior stomach in the 500 mg/kg bw/d and higher dose groups, and upon histological examination, erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema of the submucosal tissue in the 250 mg/kg bw/d and higher dose groups. On the basis of these results, the no observed effect level of the test item was judged to be less than 250 mg/kg bw/d in relation to repeated-dose toxicity in both males and females. The observed effects are limited to the forestomach and stomach of the test animals. They are not considered adverse in regard to systemic toxicity, as they are a result of the local irritation of the test item after direct contact to the mucosa due to the gavage application, during which massive amounts of the test substance are applied at once. A supplementary study was conducted to assess the no observed effect levels, which revealed a NOEL for male animals of 125 mg/kg bw/d and a NOEL for female animals of 62.5 mg/kg bw/d.

As no adverse systemic effects were observed, a NOAEL of 1000 mg/kg bw/d was determined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline study

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

No data on the substance itself is available. Therefore read across was applied to Phosphoric acid, dodecyl ester, sodium salt (CAS 50957-96-5). Besides being limited to the C12 chain length, the read across-substance differs in the cation. Both potassium (cation of the target substance) and sodium are essential alkali metal cations appearing in the human body and show very similar toxicological profiles. Toxicological effects are considered to be limited to the anionic compounds of the substances, which are similar in both the source and the target substance.

In this study conducted according to OECD Guideline 422 the read across item (Phosphoric acid, dodecyl ester, sodium salt, CAS 50957-96-5) was administered at 0 (control group), 250, 500, or 1000 mg/kg bw/d (vehicle: olive oil) to male Sprague-Dawley SPF rats for 14 days before mating, through the mating period, and up to 1 day before necropsy (42 days in total), and to female Sprague-Dawley SPF rats for 14 days before mating, through the mating and gestation periods, and up to day 4 of lactation (42 to 45 days in total), to investigate repeated-dose, reproductive and developmental toxicities. In the 0 and 1000 mg/kg groups, a 14-day recovery period was allowed after the 42-day administration period to investigate the reversibility of the toxic changes. Administration of the test substance had no effect on any of the following: general condition, results of functional tests, grip strength, spontaneous motor activity, body weight, food consumption, results of urinalysis (including water intake), or in the results of hematological or blood chemistry tests. Histopathological examinations at the end of the administration period showed indentation of the anterior stomach in the 250 mg/kg bw/d and higher dose groups, and rough mucosa or white foci in the anterior stomach in the 500 mg/kg and higher dose groups, and upon histological examination, erosions/ulcers of the anterior stomach, thickening of the anterior stomach mucosa, and edema of the submucosal tissue in the 250 mg/kg bw/d and higher dose groups. On the basis of these results, the no observed effect level of the test item was judged to be less than 250 mg/kg bw/d in relation to repeated-dose toxicity in both males and females. The observed effects are limited to the forestomach and stomach of the test animals. They are not considered adverse with regard to systemic toxicity, as they are a result of the local irritation of the test item after direct contact to the mucosa due to the gavage application, during which massive amounts of the test substance are applied at once. A supplementary study was conducted to assess the no observed effect levels, which revealed a NOEL for male animals of 125 mg/kg bw/d and a NOEL for female animals of 62.5 mg/kg bw/d.

As no adverse systemic effects were observed, a NOAEL of 1000 mg/kg bw/d was determined.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.