Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 05 January 2012 and 13 July 2012 (in-life phase: 13 January 2012 to 24 February)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Directive 96/54/EC & Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA: OPPTS 870.3050 (2000)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: METI, MHLW and MOE Guidelines of 21/11/2003.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011, histopathology examination phase performed in accordance with the Swiss Ordinance relating to GLP, adopted May 18 2005 [SR 813.112.1]

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

EC Number:

300-326-6

EC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Cas Number:

93925-25-8

Molecular formula:

Complex

IUPAC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Test material form:

liquid: viscous

Details on test material:

- Substance type: Organic
- Physical state: White liquid
- Analytical purity: Considered to be 100 % due to the UVCB character
- Lot/batch No.: 0100823714
- Expiration date of the lot/batch: 28 September 2012
- Storage condition of test material: Room temperature, in the dark
- Other: Date received at the test laboratory: 28 September 2011

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Han™:RccHan™:WIST strain animals were supplied from Harlan Laboratories U.K. Ltd., Oxon U.K.
- Age at study initiation: Approximately 6 to 8 weeks
- Weight at study initiation: Males: 201 to 232 g, females: 153 to 190 g.
- Fasting period before study: No
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, U.K.).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 8 d, during which their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 55 ± 15 % relative humidity
- Air changes: 15/h (at least)
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: First day of treatment 13 January 2012, final day of necropsy 24 February 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP grade

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in arachis oil BP. On the basis of the analytical investigations, formulations were prepared weekly during the treatment period and stored at approximately 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 0, 7.5, 75 and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw; the volume of test and control item administered to each animal was based on the most recent body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration, stability and homogeneity of the test item at Harlan Laboratories Ltd., Shardlow, U.K., Analytical Services. The results indicate that the prepared formulations were within ± 5 % of the nominal concentration and stable for at least 14 d.

Duration of treatment / exposure:

28 d (twenty-eight consecutive days)

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 300 and 1000 mg/kg bw per d; the volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.
Basis:
actual ingested

No. of animals per sex per dose:

5 (altogether 50 test animals were used, whereof 25 were males and 25 females including the highest dose recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available toxicity information and compliance with regulatory hazard classification.
- Rationale for selecting satellite groups: To enable the examination of reversibility after exposure, two recovery groups, each of 5 males and 5 females, were treated with the highest dose (1000 mg/kg bw per d) or the vehicle alone for 28 consecutive days and then maintained without treatment for a further 14 d.
- Post-exposure recovery period in satellite groups: 14 d (days 29 to 42)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 min post dosing and 1 and 5 h after dosing during the working week. Animals were observed immediately before and after dosing and 1 h after dosing at weekends. During the treatment-free period, animals were observed daily.
- Cage side observations checked: Overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on days 7, 14, 21 and 26
- Parameters examined: Signs of functional/behavioural toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill and, in the case of recovery group animals, on days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight per d: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time ∙ 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except from day 19 where water intake was measured gravimetrically. Where possible intergroup difference was detected from day 19, water consumption was continued to be measured and recorded for each cage group until the termination of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Non-recovery group animals at the end of the treatment period (day 28) and recovery group animals at the end of the treatment-free period (day 42); where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 29 and 43.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All
- Parameters checked: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (i.e. mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT) and Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Non-recovery group animals at the end of the treatment period (day 28) and recovery group animals at the end of the treatment-free period (day 42); where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 29 and 43
- Animals fasted: No
- How many animals: All
- Parameters checked: Urea Inorganic phosphorus (P), Glucose Gamma glutamyltranspeptidase (γGT), Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT), Albumin Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), total cholesterol (Chol), Calcium (Ca++), Total bilirubin (Bili) and Bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: Non-recovery group animals at the end of the treatment period (day 28) and recovery group animals at the end of the treatment-free period (day 42)
- Metabolism cages used for collection of urine: Yes; urine samples were collected overnight by housing the rats in metabolism cages.
- Animals fasted: During overnight housing in metabolism cages animals were maintained under conditions of normal hydration during collection but without access to food.
- How many animals: All
- Parameters examined: Volume, Ketones, Specific Gravity Bilirubin, pH, Urobilinogen, Protein, Blood and Glucose

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4, observations were carried out from approximately 2 h after dosing on each occasion.
- Dose groups that were examined: All
- Battery of functions tested: (1) to (3), see following paragraphs
(1) Behavioural Assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait and co-ordination, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. The test was developed from the methods used by Irwin (1968) and Moser et al (1988).
(2) Functional Performance Tests: Motor Activity and Forelimb/Hindlimb Grip Strength
Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was 1 h for each animal. The time in seconds each animal was active and mobile was recorded for the overall hour period and also during the final 20 % of the period (considered to be the asymptotic period, Reiter & MacPhail 1979).
Forelimb/Hindlimb Grip Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
(3) Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex and Finger approach.
- Irwin S (1968). Comprehensive Observational Assessment: Ia. A Systematic, Quantitative Procedure for Assessing the Behavioral and Physiologic State of the Mouse. Psychopharmacologia 13:222-57.
- Meyer OA et al (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehavioural Toxicology 1:233-6.
- Moser VC et al (1988). Comparison of Chlordimeform and Carbaryl using a Functional Observational Battery. Fundamental and Applied Toxicology 11:189-206.
- Reiter LW, MacPhail RC (1979). Motor Activity: A Survey of Methods with Potential Use in Toxicology Testing. Neurobehavioral Toxicology 1(1):53-66.

OTHER:
- Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored at approximately -20 °C.
- Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles (with coagulating glands and fluids) and Uterus with Cervix

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10 % formalin except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum) Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating, Duodenum, Epididymides, Skin (hind limb), Eyes, Spinal cord (cervical, mid thoracic and lumbar), Gross lesions, Heart, Spleen, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/Parathyroid, Lungs (with bronchi), Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland, Uterus & Cervix, Muscle (skeletal), Vagina and Oesophagus

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values were appropriate. Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p < 0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the Provantis™ Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dennett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Isolated incidences of increased salivation at 1000 mg/kg bw per d, considered not to be of toxicological importance

Mortality:

no mortality observed

Description (incidence):

Isolated incidences of increased salivation at 1000 mg/kg bw per d, considered not to be of toxicological importance

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Intergroup differences detected in erythrocyte and haematocrit, considered not to be of toxicological importance

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Intergroup differences detected in urea, cholesterol, chloride concentration, phosphorus, alkaline phosphatase and creatinine, considered not to be of toxicological importance

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Intergroup differences detected in forelimb grip strength and overall activity, considered to be of no toxicological importance

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Intergroup differences detected in pituitary and thyroid weight, considered not to be of toxicological importance

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic intergroup differences, considered not to be of toxicological importance

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Findings recorded were within the range of normal background lesions which may be recorded in rats of the strain and age used

Histopathological findings: neoplastic:

no effects observed

Details on results:

No differences between treated and control animals were observed or were considered not to be indicative of test item toxicity as explained below:

Clinical signs
- Four males treated with 1000 mg/kg bw per d showed isolated incidents of increased salivation on either days 21 or 25 and two females from this treatment group showed increased salivation on day 21 only. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

Haematology
- Males treated with 1000 mg/kg bw per d showed statistically significant increases in erythrocyte count and haematocrit. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of any associated changes the intergroup differences were considered not to be of toxicological importance.

Clinical Chemistry
- Males treated with 1000 mg/kg bw per d showed statistically significant increases in urea and cholesterol, with the effect in cholesterol also extending to 300 mg/kg bw per d males. The majority of individual values were within the normal ranges for rats of the strain and age used and in the absence of any associated histology changes the intergroup differences were considered not to be of toxicological importance.
- Recovery 1000 mg/kg bw per d males showed a statistically significant increase in chloride concentration following 14 d without treatment. All individual values were within the normal range for rats of the strain and age used and in the absence of a similar result in animals at the end of the treatment period the intergroup difference was considered not to be of toxicological significance.
- Females from all treatment groups showed a statistically significant increase in phosphorus. Females treated with 1000 and 300 mg/kg bw per d and recovery 1000 mg/kg bw per d females showed a reduction in alkaline phosphatase. All individual values were within the normal ranges for rats of the strain and age used and in the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.
- Females treated with 1000 and 300 mg/kg bw per d also showed a statistically significant increase in creatinine. The majority of individual values were within the normal range for rats of the strain and age used and in the absence of any associated histology changes the intergroup differences were considered not to be of toxicological importance.

Neurobehaviour (Functional Observations)
A. Behavioural Assessments:
- All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.
B. Functional Performance Tests:
- Males treated with 30 mg/kg bw per d showed a statistically significant increase in overall motor activity. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.
- Males treated with 1000 mg/kg bw per d showed a statistically significant increase in mean fore limb grip strength whilst females from this treatment group showed a statistically significant reduction in mean fore limb grip strength. All of the individual values were within the normal range for rats of the strain and age used and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Thyroid Hormone Assessment
No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
- Females from all treatment groups showed statistically significant reductions in thyroid weight. Females treated with 1000 mg/kg bw per d also showed a statistically significant increase in absolute and relative pituitary weight. In the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological importance.

Necropsy
- One female treated with 300 mg/kg bw per d had an enlarged and fluid filled uterus. Microscopic examination of this female revealed cornual dilation. In the absence of a similar effect detected in females treated with 1000 mg/kg bw per d the macroscopic finding was considered not to be of toxicological importance.
- One control male had small testes and epididymides. Microscopic examination revealed azoospermia in both epididymides and tubular degeneration in both testes. Observations of this nature are occasionally observed in control males and therefore in the absence of treatment are considered of no toxicological importance.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No 28 d subacute toxicity up to 1000 mg/kg bw per d, no actual NOEAL established

Executive summary:

The repeated dose toxicity of the test item by oral route was investigated in a GLP-compliant study using male and female Wistar rats according to the EU B.7 (2008), OECD TG 407 (2008) and OPPTS 870.3050 (2000) protocols and compliant to the Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc. (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

The test item was administered by gavage to three groups, each of five male and five female Wistar rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg bw per d. A control group of five males and five females was treated with vehicle alone (arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw per d) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further 14 d. Clinical signs, body weight change and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

There were no unscheduled deaths during the study and no clinically observed signs of toxicity were detected. The behavioural assessment revealed no treatment-related changes and there were no toxicologically significant changes in functional performance. The sensory reactivity and the body weight development were not different in treated animals when compared to controls and food and water consumption showed as well no intergroup differences. The haematological investigation, blood chemistry and urinalysis showed no toxicologically significant effects in the parameters examined. The organ weights were comparable between treatments and control and finally no toxicologically significant macroscopic abnormalities were detected during the Necropsy and Histopathology.

In conclusion the administration of the test item up to a dose of 1000 mg/kg bw per d, did not result in any toxicologically significant effects. No actual NOAEL could be established as no effects up to the maximum dose level recommended by the test guidance was recorded. On the basis of this study no indication for classification is given.