Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 07, 2016 to December 19, 2016

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:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Version / remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3050

Version / remarks:

Repeated Dose 28-day oral toxicity study in rodents, July 2000.

Deviations:

yes

Remarks:

but were considered not to have affected the outcome or the achievement of the study objectives

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity (dry matter): 10.0 % (taken into account for dose calculations)
Appearance: whitish liquid

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI (Han)

Details on species / strain selection:

One of the rodent species acceptable to the regulatory authorities. Historical control data for the strain are available at the Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species/strain: Wistar rats: Crl: WI (Han).
Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint Germain sur l'Arbresle, France.
Acclimatisation period: 9 days between animal arrival and the start of treatment.
Number of animals in the study: 40 males and 40 females.
Age at initiation of treatment: Virgin males: approximately 9 weeks; Virgin females: approximately 8 weeks.
Body weight range at initiation of treatment: Virgin males: 307 to 341 g; Virgin females: 167 to 201 g.
Housing: One air-conditioned room in a barrier protected unit.
Temperature: 22 +/- 3 °C.
Relative humidity: > 35 %.
Air changes: At least 10 air changes per hour.
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts).
Diet: Rat pelleted complete diet ad libitum (Diet reference A04C-10)
Water: Softened and filtered (0.2 µm) mains drinking water was available ad libitum
Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages meeting European directive 2010/63/EU requirements.

Route of administration:

oral: gavage

Details on route of administration:

Method of administration: Oral gavage using a plastic cannula.
Volume of administration: 10 mL/kg/day.
Individual dose volumes were calculated using the latest body weight.
(Rationale for choice of route of administration: oral route was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.)

Vehicle:

water

Details on oral exposure:

The test substance was prepared as a solution in the vehicle according to Standard Operating Procedures of the Test Facility at concentrations 3, 10 and 30 mg/g. Additional formulations were performed at concentrations of 1 and 77 mg/g for stability analysis purpose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Quadruple accurately measured 0.5 g samples were taken from each formulation, used on the first day of treatment immediately after preparation (Time 0) and after 6 hours (Time T+6) stored at ambient temperature. The samples were then stored frozen (between -25 and -15 °C).
- Analysis of the samples (Charles River Laboratories Den Bosch BV. project 515269) was performed at the Test Site according to a validated method (Charles River Laboratories Den Bosch BV. project 514913).
- Formulations at the entire range were stable when stored in a freezer (≤ -15°C) for at least 7 days. Therefore, the stability under sample shipment conditions was demonstrated. The accuracy of preparation and homogeneity of the test substance in formulations was determined.
The concentrations analysed in the formulations of 30, 100 and 300 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the control group formulations. The formulations of 30 and 300 mg/kg bw/day were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

For males: 14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 30 days).
For females: from 14 days before mating to Day 3 of lactation. During gestation (the first day of gestation was designated as G 0) and during lactation (the first day of birth is designated as L 0). Apparently one unmated female was treated 29 days after the last day of the mating period (i.e. 54 days). Females that fail to produce a viable litter were treated up to Day 25 of gestation.

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 0 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 3 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 10 mg/g

Dose / conc.:

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

Remarks:

Dose volume: 10 mL/kg/day
Dose concentration: 30 mg/g

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Three groups of 10 male and 10 female Wistar Han rats were given the test substance, by daily oral (gavage) administration at dose levels of 30, 100 and 300 mg/kg bw/day from 14 days before mating (Day 1) until Day 30 for males or until Day 3 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 10 mL/kg/day of the vehicle (water for injection).
- Clinical condition, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre-mating period (Day 14). After two weeks of treatment, one male and one female of the same group were paired for a maximum of 10 days. The females were allowed to give birth and litter parameters, including the number of pups born, pup survival, sex and pup weights were recorded up to postnatal Day 4. Functional tests were performed at the end of the treatment period (Day 30) for 5 selected males in each group and on Day 3 of lactation for 5 selected females in each group. The males were necropsied after the pairing period. The dams and pups were necropsied on Day 4 of lactation. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from any dead animal, from half of the group 30 and 300 mg/kg bw/day animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups.

Positive control:

-

Observations and examinations performed and frequency:

- Morbidity/Mortality: Adult animals were observed at least twice daily (i.e. at the beginning and at the end of the working day, including weekends and public holidays) to detect any which were dead.

- Clinical signs: All animals were observed daily for clinical signs. To detect any clinical signs or reactions to treatment, the animals were observed before and at least once after dosing. A full clinical examination was performed on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.

- Body weight: Individual body weights for male were recorded weekly. Individual body weights for female were recorded as follows:
weekly during pre-mating and mating periods (only pre-mating data are reported)
on Days 0, 6, 9, 12, 15, 18 and 20 of gestation
on Days 1 and 4 of lactation.

- Food consumption: Food consumption of each male was recorded weekly during the pre-mating period.Food consumption of each female was recorded for the following periods:
weekly during the pre-mating period
on Days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 of gestation
on Days 1 to 4 of lactation.

- Functional tests: Animals examined: 5 randomly selected animals/sex/group (for females, only those with live pups were selected). Frequency:
Males: at the end of the treatment period, shortly before sacrifice (Day 30).
Females: during lactation, shortly before scheduled sacrifice (Day 3 of lactation).
The following tests were performed: auditory reflex, pupillary reflex, righting reflex, fore- and hind-limb grip strength, locomotor activity in an open-field test.

- Blood collection
Animals examined and frequency: Five randomly selected animals/sex/group at the end of the pre-mating period (Day 14).
Method of blood sampling: Blood was withdrawn from the retro-orbital sinus under isoflurane anaesthesia from animals fasted for at least 16 hours. Samples were collected (before daily dosing): in tubes containing K2-EDTA for haematology parameters, in tubes containing trisodium citrate for coagulation parameters, in tubes without anticoagulant for clinical chemistry parameters.
1. Haematology: Red blood cell count, Haemoglobin, Packed cell volume, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Reticulocyte count, Platelet count, Total white blood cell count, Differential white blood cell count.
2. Coagulation: Prothrombin time, Activated partial thromboplastin time, Fibrinogen.
3. Serum clinical chemistry: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Total cholesterol, Total bilirubin, Total protein, Albumin, Creatinine, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase, Bile acids.

Sacrifice and pathology:

- At the end of the treatment period, all rats were necropsied. A limited list of tissues was examined histologically from rats in the groups 30 and 300 mg/kg bw/day at the end of the treatment period, as well as gross lesions from all the other rats.
All adult males and females were killed by carbon dioxide inhalation, exsanguinated and then submitted to a macroscopic examination with special attention being paid to the reproductive organs:
1. males: after completion of the mating period, corresponding to a minimum of 28 days of oral administration
2. females: on day 4 of lactation. Females that failed to produce a viable litter by day 26 post coitum were killed and necropsied
- The organs were weighed at scheduled necropsy for the 5 randomly selected adult males and females of each group (for females, only those with live pups). For the other 5 males of each group, only epididymides and testes were weighed. Paired organs were weighed together. The organs were weighed after dissection of fat and other contiguous tissues. Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight).
- The organs/tissues were sampled for the 5 randomly selected adult males and females of each group (only females with live pups) and any animal found dead. Only a limited list of organs/tissues was sampled for the remaining males and females, including females that failed to produce a viable litter by day 26 post-coitum (unmated females or mated females that did not deliver after 26 days post-coitum) or females with total litter death. All organs/tissues sampled were fixed and preserved in 10% neutral formalin except for Harderian glands, testes, epididymides, eyes and optic nerves which were fixed in modified Davidson's fluid. The same sampling and trimming procedures was used for all applicable tissues in all applicable dose groups. Paraffin-embedded blocks were prepared only for tissues which were evaluated histopathologically.
Histopathology:
Histological evaluation was performed as follows: for all organs/tissues from all animals found dead during the study, for all macroscopic lesions from all dose group animals , for all organs/tissues from the 5 randomly selected adult males and females of groups control and 300 mg/kg bw/day (for females, only those with live pups), for the testes from the 5 randomly selected adult males of groups control and 300 mg/kg bw/day and all males suspected to be infertile.
A detailed qualitative evaluation of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings were recorded, where appropriate: for the reproductive organs of all males that fail to sire and all females that fail to deliver healthy pups and the liver, thyroid glands from group 2 and 3 males and females, and the uterus from group 2 and 3 females.
- Histological slides were prepared and stained with haematoxylin and eosin (HE). In addition, Periodic Acid Schiff-(PAS)-stained sections were prepared and examined for the testes.
- Severity grades were assigned to non-neoplastic histologic diagnoses, using a theoretical maximum range of grades.

Other examinations:

-

Statistics:

Statistical analyses were performed by the Provantis data acquisition system and analysed using a SAS software package

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed in the 300 mg/kg bw/day group consisted of decreased activity, modified feaces, stereotypic behaviour and/or piloerection. Compared with the concurrent controls, there was an increased incidence of animals showing hypersalivation associated or not with abnormal foraging and/or pedaling in all test substance groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related decreased body weight gains were associated with lower mean body weights at 300 mg/kg bw/day in both males and females throughout the treatment period. Minor differences noted in mean body weight gain in the 100 mg/kg bw/day group were not considered to be toxicologically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related decreased food consumption were associated with lower mean body weights at 300 mg/kg/day in both males and females throughout the treatment period. Minor differences noted in food consumption in the 100 mg/kg/day group were not considered to be toxicologically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were dose-related haematological (decreased red blood cell and reticulocyte counts, prolonged APTT, decreased fibrinogen) in test substance groups. These changes were considered secondary to the effects on food consumption, and/or on the liver and not adverse given their magnitudes at 30 and 100 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were dose-related serum clinical chemistry changes (cholesterol levels, increases in Cl, Ca and urea, and slightly increased ALP, AST and ALT levels) in test substance groups. These changes were considered secondary to the effects on food consumption, and/or on the liver and not adverse given their magnitudes at 30 and 100 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no obvious effect of treatment on the auditory, pupillary and righting reflexes or gripping and open-field tests for the males or females at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weight at necropsy in males and females given 300 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale/enlarged appearance of liver at necropsy in males and females given 300 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related pathological changes (non-adverse) observed in the liver and thyroid glands consisted of:
- In the liver, minimal to moderate peri-portal to diffuse hepatocellular glycogen deposits were noted at 100 and 300 mg/kg bw/day in both sexes. Minimal centrilobular hepatocellular hypertrophy, also considered likely treatment-related, was observed in two females given 300 mg/kg bw/day. These findings correlated with pale/enlarged appearance and higher liver weight at necropsy in males and females given 300 mg/kg bw/day.
- In the thyroid glands, minimal follicular cell hypertrophy was observed in one male and 2 females treated at 300 mg/kg bw/day and one male and one female at 30 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Conclusions:

Under the study conditions, the systemic sub-acute NOAEL of the substance in rats was determined to be 100 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance according to OECD Guideline 422, EU Method B.7, US EPA OPPTS 870.3650 and 870.3050, in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats (10 males and10 females per dose group) from 14 d before mating, during mating, during organogenesis and until Day 3 of lactation (females) at doses of 0, 30, 100 and 300 mg/kg bw/day. Mortality, clinical signs, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre-mating period (Day 14). Functional tests were performed at the end of the treatment period (Day 30) for 5 selected males in each group and on Day 3 of lactation for 5 selected females in each group. The males were necropsied after the pairing period. The dams and pups were necropsied on Day 4 of lactation. All animals were submitted to a macroscopic examination, selected organs were weighed and major organs were examined microscopically. There was no test substance-related mortality in any group. Clinical signs observed at the high dose consisted of decreased activity, stereotypic behaviour and/or piloerection. Parental toxicity at 300 mg/kg bw/day consisted of decreased body weight, body weight gains and food consumption, as well as clinical signs at 300 mg/kg bw/day, dose-related haematological and serum clinical chemistry changes such as decreased red blood cell and reticulocyte counts, prolonged APTT, decreased fibrinogen and cholesterol levels, increases in Cl, Ca and urea, and slightly increased ALP, ASAT and ALAT levels. These changes were considered secondary to the effects on food consumption, and/or on the liver and not adverse given their magnitudes at 30 and 100 mg/kg bw/day. Non-adverse pathological changes were observed in the liver (minimal centrilobular hepatocellular hypertrophy in females at 300 mg/kg bw/day and minimal to moderate peri-portal to diffuse hepatocellular glycogen-like deposits at 100 and 300 mg/kg bw/day) and thyroid glands (minimal follicular cell hypertrophy at 30 and 300 mg/kg bw/day). Under the study conditions, the sub-acute systemic NOAEL of the substance in rats was determined to be 100 mg/kg/day (Pique, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the repeated dose toxicity of the substance according to OECD Guideline 422, EU Method B.7, US EPA OPPTS 870.3650 and 870.3050, in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats (10 males and10 females per dose group) from 14 d before mating, during mating, during organogenesis and until Day 3 of lactation (females) at doses of 0, 30, 100 and 300 mg/kg bw/day. Mortality, clinical signs, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre-mating period (Day 14). Functional tests were performed at the end of the treatment period (Day 30) for 5 selected males in each group and on Day 3 of lactation for 5 selected females in each group. The males were necropsied after the pairing period. The dams and pups were necropsied on Day 4 of lactation. All animals were submitted to a macroscopic examination, selected organs were weighed and major organs were examined microscopically. There was no test substance-related mortality in any group. Clinical signs observed at the high dose consisted of decreased activity, stereotypic behaviour and/or piloerection. Parental toxicity at 300 mg/kg bw/day consisted of decreased body weight, body weight gains and food consumption, as well as clinical signs at 300 mg/kg bw/day, dose-related haematological and serum clinical chemistry changes such as decreased red blood cell and reticulocyte counts, prolonged APTT, decreased fibrinogen and cholesterol levels, increases in Cl, Ca and urea, and slightly increased ALP, ASAT and ALAT levels. These changes were considered secondary to the effects on food consumption, and/or on the liver and not adverse given their magnitudes at 30 and 100 mg/kg bw/day. Non-adverse pathological changes were observed in the liver (minimal centrilobular hepatocellular hypertrophy in females at 300 mg/kg bw/day and minimal to moderate peri-portal to diffuse hepatocellular glycogen-like deposits at 100 and 300 mg/kg bw/day) and thyroid glands (minimal follicular cell hypertrophy at 30 and 300 mg/kg bw/day). Under the study conditions, the sub-acute systemic NOAEL of the substance in rats was determined to be 100 mg/kg/day (Pique, 2017).

Justification for classification or non-classification

Based on the combined repeat dose-reproductive toxicity screening study with the substance, no classification for repeated dose toxicity is warranted according to EU CLP (EC 1272/2008) criteria.