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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
The critical effect observed in the combined repeated dose and reproduction / developmental screening is due to local toxicity linked to a dose-concentration independantly to a duration of exposure.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August - 21 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in the report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Phosphonic acid (Phosphorous acid)
- Substance type: White crystalline solid
- Physical state: Solid
- Purity: See Certificate of Analysis
- Lot/batch No.: PAFK07F002
- Expiration date of the lot/batch: 31 July 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark desiccated
- pH: <1 at concentration of 1%
- Purity/composition correction factor required: No
- Hygroscopic: Yes, store desiccated
- Volatile: No
- Stability at higher temperatures: Yes, maximum temperature: <150°C
- Solubility in vehicle (water): >1067 g/L

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks (Groups 1-4) and 12 weeks (additional animals for Group 4).
- Weight at study initiation: mean weight at start of treatment was 324 - 330 gr (males) or 211 - 214 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 31 August to 21 November 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Preventative measures: The pH of the dose preparations for Groups 2-4 was relatively low. To avoid possible local esophageal effects, the following preventative measures were taken:
1 Wiping of the flexible catheter prior to dosing of each animal.
2 From 16 October 2012 onwards, additional rinsing of the flexible catheter with approximately 0.5-0.6 mL water (Elix, Millipore S.A.S., Molsheim, France) after dosing each animal with the test formulations.

One Group 2 female was dosed according to her Day 20 post-coitum body weight, while she had just delivered her litter (which was not completely cleaned yet). The slightly higher dose volume resulted in a dose level of 103 mg/kg instead of 80 mg/kg.
Evaluation: Handling of this animal did not disturb maternal care as all pups survived until planned necropsy and no findings were noted. A slightly higher dose level for just one day of treatment will not affect data of this animal.

From 12 November 2012 onwards, it was not noted in the raw data if the flexible catheter was wiped prior to dosing of each animal.
Evaluation: In the unlikely event that this preventative action was not taken, no local esophageal effects were caused as no esophageal abnormalities were seen at necropsy.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis.

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at TNO Triskelion, The Netherlands.

Formulation samples were not transferred to the weighing room, but were dispatched directly to the test site.
Evaluation: Samples were received in good order by the test site.

Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).Two Group 1 females, three Group 2 females, one Group 3 female and one Group 4 female were not dosed during littering.
Frequency of treatment:
Once daily, 7 d/w
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 80, 250 and 750 mg/kg bw/day. During the first four days high dose animals were dosed at 750 mg/kg however due to unexpected severe toxicity the dose level was lowered to 500 mg/kg from Day 5 onwards.
Basis:
actual ingested
No. of animals per sex per dose:
Group 1-3: 10/sex
Group 4: 13 males and 11 females. (one female and three males lost during the first 5 days of treatment were replaced by spare animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study (See attached results).

Based on the results of this range finding study, dose levels for the main study were: 0, 80, 250 and 750 mg/kg.
Since no clear peak effect of occurrence of clinical signs was observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

During the first four days of the treatment period, Group 4 animals were dosed at 750 mg/kg. From Day 5 onwards, they were dosed at 500 mg/kg.

- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least immediately after dosing (no clear peak effect of occurence of clinical signs in the dose range finding study). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Sacrifice and pathology:
GROSS PATHOLOGY
All animals except for non-selected females were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and testes

Inadvertently, the terminal body weights of two Group 1 females were not determined at necropsy, and inadvertently organ weights were collected from two Group 4 males when they were not required.
Evaluation: Sufficient data was available for evaluation. Moreover, as it concerned two control animals historical control data could be taken into account. The extra organ weights were taken from animals who had been fasted and are thus representative. These data are reported.

HISTOPATHOLOGY
- According to test guidelines

By error, the thyroid of one control Female was not weighed at necropsy and a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data was available for evaluation.

Inadvertently, the adrenals, aorta, clitoral gland, thymus, liver and lungs of one Group 4 female were fixated a few minutes in Modified Davidson instead of 10% buffered formalin.
Evaluation: This did not affect the quality of the slides.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

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):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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:
MORTALITY
There were three males and one female dosed at 750 mg/kg that were euthanized in extremis or died spontaneously after 2-4 days of treatment. The dose level was subsequently lowered to 500 mg/kg. After that time, there were an additional four females and one male that died or were euthanized in extremis. The females died or were euthanized after 29-36 days and the male was killed in extremis after 9 treatment days.

CLINICAL SIGNS
At 750/500 mg/kg laboured respiration, rales, gasping, piloerection, chromodacryorrhoea of the snout, lethargy, hunched posture, salivation, hypothermia and lean appearance were noted for the animals that died or were euthanized in extremis.

Animals at 750/500 mg/kg that survived to the end of the treatment period were noted with rales, salivation, hunched posture, laboured respiration, piloerection, lean appearance, lethargy, spasms and flat posture.

Piloerection, rales, chromodacryorrhoea of the snout, hunched posture, laboured respiration and salivation were noted transiently for a few animals at 250 mg/kg. As these signs were transient and noted for a limited number of animals at this dose level, they were not considered to be adverse. Scabs on the right flank was noted for one male at 250 mg/kg, this was incidental.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
Body weights and body weight gains were lower for males at 750/500 mg/kg than controls from Day 8 of the premating period through the entire mating period (not always statistically significant). Body weights and gains were lower for females at 750/500 mg/kg than controls from Days 14-20 of the post coitum period (only absolute body weight on Day 14 was significantly lower than controls). The difference for females was mostly attributed to weight loss and reduced gains for two Group 4 females (one Group 4 female also contributed to this on post coitum Day 14; she was killed in extremis after this point).

FOOD CONSUMPTION
At 750/500 mg/kg, a trend towards slightly lower food consumption (absolute and relative to body weight) was noted for males and females during the premating period (not statistically significant). Absolute and relative food consumption was significantly lower for high dose females from Days 4-17 of the post coitum period.

Absolute food consumption was also significantly lower for females at 250 mg/kg from Days 7-11 post coitum, though the effect was only transient, and in the absence of corresponding effects on body weights, it was not considered to be adverse.

The significantly lower food consumption (absolute and relative) noted for females at 80 mg/kg from Days 0-7 and for females at 250 mg/kg from Days 0-4 of the post coitum period, respectively, were not considered to be toxicologically relevant as they occurred in the absence of a dose-related distribution.

HAEMATOLOGY
There were no toxicologically relevant effects on haematology parameters up to 750/500 mg/kg.

Haemoglobin and haematocrit values were significantly lower for males at 750/500 mg/kg and significantly higher for females at 750/500 mg/kg than controls. These were not considered to be toxicologically relevant, however, as these changes were opposite for males and females and the values remained within the range of normal biological variation and did not show a dose-dependent distribution.

CLINICAL BIOCHEMISTRY
At 750/500 mg/kg significantly lower values for total protein (males) and albumin (females) and a higher concentration of bile acids (males) compared to controls were noted.

The remaining statistically significant changes noted in this study were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes included increased concentrations of sodium (males at 250 mg/kg), chloride (males at 80, 250 and 750/500 mg/kg), and lower concentrations of urea (females at 250 mg/kg), sodium (females at 80 and 750/500 mg/kg) and inorganic phosphates (females at 250 and 750/500 mg/kg).

MACROSCOPIC EXAMINATION
Macroscopic findings noted for the animals at 750/500 mg/kg that died or were euthanized in extremis included isolated or several reddish or dark red foci on the stomach glandular mucosa, irregular surface or isolated dark red or black foci of the forestomach and/or glandular mucosa, perforations of the forestomach, fibrin-like coating on the diaphragm, thickened limiting ridge of the stomach, reduced size of spleen and thymus, enlarged lungs, GI-tract distended with gas, beginning autolysis, greenish discoloration of the lungs, pelvic dilation of the kidney, emaciated appearance, uterus contains live fetuses (one Group 4 female) and many dark red foci on the thymus.

There were no treatment-related macroscopic findings in the animals surviving to their scheduled necropsy dates.

Incidental findings noted for control and/or treated animals included enlarged or reddish discolored mesenteric lymph node, enlarged mandibular lymph node, isolated reddish foci on the thymus, reduced size of the left seminal vesicle, scabbing on the right flank, isolated tan or dark red focus/foci on one or both clitoral glands, several yellowish, soft nodules on the clitoral glands, yellowish, soft nodule on the epididymis, pelvic dilation of the kidneys and yellowish, soft nodule on the liver. These findings remained within the background range encountered among rats of this age and strain, did not show a dose-related incidence trend and were thus not considered to be treatment related or toxicologically relevant.

ORGAN WEIGHTS
At 750/500 mg/kg females had lower absolute and relative uterine weights and lower absolute spleen weights. Females at 250 mg/kg had lower absolute spleen weights as well. The lower spleen weights at 750/500 mg/kg parallel the decrease in hemopoietic foci seen in females at the microscopic examination.

MICROSCOPIC EXAMINATION
There were two Group 4 males and one Group 4 female found dead. The two males had macroscopic perforation of the forestomach, which for one Group 4 male correlated to ulceration of the forestomach. For another Group 4 male the autolysis of the stomach was too advanced for a thorough microscopic evaluation, though the presence of a few mucosal erosions still could be noted. For one Group 4 female complete ulceration of the trachea epithelium was the cause of death.
In addition, there were two Group 4 males and four Group 4 females sacrificed moribund. For one Group 4 male the cause of moribundity was marked necrosis of the trachea epithelium, for another Group 4 male no definite cause of moribundity could be established. For one Group 4 female ulceration of the glandular stomach and for another Group 4 female moderate ulceration of the trachea and bronchial epithelium were the cause of moribundity. For two Group 4 females no definite cause of moribundity could be established. These deaths are considered test item related.

There were treatment-related microscopic findings present in rats surviving to their scheduled necropsy dates in the:

Stomach
- Glandular inflammation, lymphogranulocytic was present in male rats treated at 750/500 mg/kg (4/5 rats) up to a slight degree.
- Glandular stomach erosion was present in male rats treated at 750/500 mg/kg (1/5) at a minimal degree.
- Hyperplasia of the forestomach squamous epithelium was present in male rats treated at 750/500 mg/kg (2/5) up to slight degree.
- Vacuolation of the limiting ridge was present at increased severity in male rats treated at 750/500 mg/kg (2/5) at slight degree. Minimal degrees were present in control, 80 and 250 mg/kg treated rats (all 1/5 rats) and this was considered to be the background level.
- Thickened limiting ridge was present in male rats treated at 750/500 mg/kg (2/5) up to moderate degree.

Spleen
- Hemopoietic foci, primarily erythropoiesis showed a slight decrease in 750/500 mg/kg treated female rats.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Effect levels

Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

No test substance was detected in the Group 1 formulations. The concentrations analyzed in the formulations of Group 2, 3, 4 and 4/5 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

 

The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation = 10%).

 

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference = 10%).

Applicant's summary and conclusion

Conclusions:
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 250 mg/kg (both for local and systemic toxicity).
Reproduction NOAEL: at least 500 mg/kg. As 750 mg/kg was only dosed for four days, this NOAEL could only be set at 500 mg/kg.
Developmental NOAEL: at least 250 mg/kg.
The likelihood that this NOAEL is actually higher cannot be excluded. However, the number of litters (n=6) available at the higher dose level of 750/500 mg/kg was too low to ensure a thorough evaluation of possible developmental effects.
Executive summary:

In an oral 28-d combined repeated dose toxicit/reproscreening study, which was performed in accordance with OECD Guidance 422 and under GLP conditions, four groups of ten male and ten female Wistar Han rats (acclimatized) were exposed by oral gavage to Phosphonic acid at 0 (vehicle), 80, 250 and 750/500 mg/kg bw/day. After 2 -4 days three males and one female died or were killed in extremis. These animals were replaced by spare rats. From day 5 onwards, the high dose level was adjusted from 750 to 500 mg/kg bw/day. One additional male was killed in extremis after 9 days and 4 additional females died or were euthanized after 29 -36 days.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination.

Females were exposed for 41-48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

At 750/500 mg/kg toxicity was characterized by mortality, lower body weights and food consumption, effects on clinical biochemistry parameters, macroscopic and microscopic findings of the stomach indicative of severe irritation and corrosion, and weight changes and microscopic findings of the spleen in females. While most of the effects (including mortality) were secondary to the severe localized stomach effects, the cause of moribundity could not be determined for three animals who died before the end of the scheduled treatment period. As such, a systemic effect of the test substance cannot be excluded.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:Parental NOAEL: 250 mg/kg bw/day.