Diethyl (hydroxymethyl)phosphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and appropriate guidelines

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Histology and light microscopy was conducted on the uterus and cervix only, while the protocol also mentioned the oviducts.

Principles of method if other than guideline:

n/a

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl (hydroxymethyl)phosphonate

EC Number:

221-391-6

EC Name:

Diethyl (hydroxymethyl)phosphonate

Cas Number:

3084-40-0

Molecular formula:

C5H13O4P

IUPAC Name:

diethyl (hydroxymethyl)phosphonate

Test material form:

other: meduim viscosity liquid

Details on test material:

E06-16 (Lot# 1041-37-6, Sample: T# 207), transparent "medium-viscoosity" liquid.

Specific details on test material used for the study:

Lot number 540-1013-14

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Supplier Charles River (UK) Ltd. The rat (virgin) was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals ordered: 44 males and 44 females.
Duration of acclimatisation: Six days before commencement of treatment.
Age of animals at the start of the study: Approximately 10 weeks old.
Diet supply: Diet SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Water supply: Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Weight range of animals at the start of the study: Males: 330 to 375 g; Females. 201 to 254 g
Identification: Each adult animal was asigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment.
Offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 19-23ºC and 40-70%.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Number of animals per cage: Pre-pairing up to five animals
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

corn oil

Remarks:

Batch numbers - L24105 and L24287/AF) supplied by Consumer’s Pride.

Details on oral exposure:

Gavage, using a suitably graduated syringe and a Nelaton catheter inserted via the mouth.
Treated at Constant doses in mg/kg. Volume dose 5 mL/kg body weight.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of a homogenous suspension of the test substance in the vehicle at concentrations of 5 mg/mL (6 mg/ml including correction factor) and 200 mg/mL (240 mg/ml including correction factor) was analyzed over a period of up to 15 days.
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of acetone. The extract was diluted using acetone, to provide a solution containing E06-16 at an expected concentration of 100 μg/mL. Internal standard (100 μL) was added to each sample solution (1 mL) and vortex mixed prior to analysis. The concentration of E06-16 in the final solution was quantified by GC using FID detection.

Duration of treatment / exposure:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Therefore, it was considered that the animals were treated for at least 28 days. The F1 generation received no direct administration of the test substance, E06-16. Any exposure to the test substance or metabolites was through the mother to the offspring in utero and/or through the milk.

Frequency of treatment:

Once daily, at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One Control group and three treated groups (100, 300, 1000 mg/kg bw) of animals.
Each group was comprising of 10 male and 10 female rats.

Control animals:

yes, concurrent vehicle

Details on study design:

1. Three groups comprising of ten male and ten female rats received E06-16 at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. A similarly constituted Control group received the vehicle, corn oil.

2. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females which littered were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

3. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

4. The clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight
and macropathology for all offspring were also assessed.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the
occupant(s). Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate. During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.

Sacrifice and pathology:

All adult animals were subject to a detailed necropsy. After a review of the history of each
animal, a full macroscopic examination of the tissues was performed. All external features
and orifices were examined visually. Any abnormality in the appearance or size of any organ
and tissue (external and cut surface) was recorded and the required tissue samples preserved
in appropriate fixative.

Other examinations:

The study included also evaluation of the product's reproduction and developmental toxicity. Thus, oestrous cycles, pre-coital interval, mating performance, fertility and gestation length were undertaken, and the clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight and macropathology for all offspring were assessed.

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine examination that could be associated to treatment and no signs were observed in association with dose administration.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight and bodyweight gain for males was considered unaffected by treatment when compared with Controls. The occasional slight variations seenin the femaees were considered not adverse and unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No effect that could be attributed to treatment on food consumption for males and females receiving 100, 300 or 1000 mg/kg/day, when compared to Controls.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect on food efficiency was observed.

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

not specified

Description (incidence and severity):

n/a

Ophthalmological findings:

not specified

Description (incidence and severity):

not specified but no other effect that could be associated to treatment was observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effect that could be attributed to treatment on clinical condition were observed. The haematological examination of the blood in Week 2 of treatment which revealled high absolute reticulocyte and platelet counts in males treated at 1000 mg/kg/day, considered non treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effect that could be attributed to treatment on biochemistry was observed. The biochemical changes that were seen in the treated animals were considered non treatment related.

Urinalysis findings:

not examined

Description (incidence and severity):

n/a

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Motor activity was unaffected for males and females receiving E06-16 up to 1000 mg/kg/day, when compared to Controls. There were no clear effects on either rearing (high beam) or ambulatory (low beam) motor activity for males or females and total activity was similar across all groups for both sexes.

Immunological findings:

not specified

Description (incidence and severity):

n/a

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

the effects on liver's weight gaining seen in males were considered an adaptive, non-adverse response. All other organ weights were similar to Controls and considered unaffected by treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The incidence and distribution of all findings were consistent with the common background macroscopic findings seen in Sprague-Dawley rats at these laboratories.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sensory reactivity and grip strength was unaffected by treatment. The few changes that were observed in males were within the expected range determined from background control data and considered not related to treatment. Females were unaffected.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Changes related to treatment with E06-16 were seen in the liver and thyroids, however, their occurrence in groups was considered to be within the common background incidence seen at these laboratories.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A single incidence of lymphoma was observed in one animal, which was considered to be within the common background incidence of this finding seen at these laboratories and unrelated to treatment. The incidence and distribution of all other findings were consistent with the common
background of microscopic findings seen in Sprague-Dawley rats at these laboratories.

Other effects:

no effects observed

Description (incidence and severity):

No evidence for adverse general systemic toxicity or effects on reproductive performance/offspring development attributed to treatment were observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

other: effects on reproductive performance/offspring development

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

n/a

Applicant's summary and conclusion

Conclusions:

It was concluded that in the absence of any evidence for adverse general systemic toxicity or effects on reproductive performance/offspring development that the NOAEL
(no-observed-adverse-effect-level) was 1000 mg/kg/day.

Executive summary:

The objective of this study was the assessment of general systemic toxic potential of E06-16 in rats, including a screen for reproductive/development effects, with administration of the test substance by the oral gavage route for at least four weeks.

Three groups comprising of ten male and ten female rats received E06-16 at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks.

Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females which littered were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, ano-genital distance, body weight

and macropathology for all offspring were also assessed.