[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-07-23 to 2013-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Cross-reference 3

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached document justification

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Dose descriptor:

NOAEL

Effect level:

> 10 - < 20 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

, seeking for additional information: (i) recovery groups to evaluate the reversibility of liver effects, and (ii) specific assessment of effects on the reporductive cycle.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

2012-11-30

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea

IUPAC Name:

Tetrakis(hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea

Constituent 2

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

EC Number:

500-057-6

EC Name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Cas Number:

27104-30-9

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea

Test material form:

other: clear yellow liquid

Details on test material:

- Name of test material (as cited in study report): Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea
- Physical state:clear yellow liquid
- Storage condition of test material: room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: approximately six to eight weeks old
- Weight at study initiation: the males weighed 165 to 196g, the females weighed 135 to 174g
- Fasting period before study: No
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK)
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage
- Acclimation period: The animals were acclimatised for seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From2012-08-17 To 2012-12-20

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water. Results from the stability and homogeneity show the formulations to be stable for at least four hours. Formulations were therefore prepared daily.
VEHICLE
- Concentration in vehicle: 0, 6.54, 13.1 and 39.2 mg/kg (Active ingredient)
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Tetrakis(hydroxymethyl)phosphonium chloride, oligomeric reaction products with urea (CAS#27104-30-9) in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples: The test item formulations were diluted with water to give final, theoretical test item concentrations of approximately 0.3 mg/ml.
Standards: Standard solutions of test item were prepared in water at a nominal concentration of 0.3 mg/ml.

The standard and sample solutions were analysed by HPLC using the following conditions:
+ HPLC : Agilent Technologies 1050 or 1200, incorporating autosampler and workstation
+ Column : Synergi Hydro RP 4μm (250 x 4.6 mm id)
+ Mobile phase : 2.2g of sodium dihydrogen orthophosphate in 1 litre of water, pH adjusted to 6 with sodium hydroxide
+ Flow-rate : 1 ml/min
+ UV detector wavelength: 205 nm
+ Injection volume : 25 μl
+ Retention time : ~ 6.8 mins

Homogeneity Determinations: The homogeneity determinations at higher levels were performed under Harlan Laboratories Ltd. project number 2897-0002. The test item formulations at approximately 0.3 mg/ml were deemed to be homogeneous by visual inspection.

Stability Determinations: The stability determinations at higher levels were performed under Harlan Laboratories Ltd. project number 2897-0002. The test item formulations at approximately 0.3 mg/ml were sampled and analysed initially and then after storage at ambient temperature for four hours.

Verification of Test Item Formulation Concentrations: The test item formulations were sampled within four hours of preparation.

Linearity: The linearity determinations were performed under Harlan Laboratories Ltd. project number 2897-0002

Specificity: Analysis of the solvent and a blank Distilled Water (control) produced no signal that interfered with the signal due to the test item

Accuracy: The accuracy determinations at higher levels were performed under Harlan Laboratories Ltd. project number 2897-0002. The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion: The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study

Duration of treatment / exposure:

ninety consecutive days

Frequency of treatment:

once a day (7 days/week)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 10, 20 and 60 mg/kg Test item
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 6.54, 13.1, 39.2 mg/kg active ingredient
Basis:
actual ingested

No. of animals per sex per dose:

Control:20 males and 20 females (10 control and 10 recovery control)
6.54 mg/kg and 13.1 mg/kg AI: 10 males and 10 females per group.
39.2 mg/kg AI: 20 males and 20 females (10 main group and 10 recovery group)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: On the basis of previous toxicity data (acute toxicity and repeated dose toxicity, Rep. Tox. Oral 28-day V1 2010 HARL)
- Rationale for selecting satellite groups: (based on the observation during the 28 days study)
- Post-exposure recovery period in satellite groups: yes: for 28 days

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:first day of dosing: before treatment, 30 minutes, 1h and 5h after dosing. All the other day: before and 1h after dosing. During the recovery period: daily
- Cage side observations were performed. The following parameters were observed:
Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal and Tail elevation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. During Week 12 functional performances tests were also performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.
The following test were performed:
- Behavioural Assessments: Detailed individual clinical observations were performed for each non-recovery animal
- Functional Performance Tests: Animals of one sex from each non-recovery dose group were tested at each occasion
- Forelimb/Hindlimb Grip Strength: Each non-recovery animal
- Sensory Reactivity: Each non-recovery animal

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment and before termination of treatment (during Week 12)
- Dose groups that were examined: control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 90 for non recovery animal and day 118 for recovery animals
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all non-recovery animals from each test and control group at the end of the study and on all recovery group animals at the end of the treatment-free period
- Following parameters were examined:
+ Haemoglobin (Hb)
+ Erythrocyte count (RBC)
+ Haematocrit (Hct)
+ Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
+ Total leucocyte count (WBC)
+ Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
+ Platelet count (PLT)
+Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
+ Prothrombin time (CT) was assessed by ‘Innovin’
+ Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 90 for non recovery animal and day 118 for recovery animals
- Animals fasted: No
- How many animals: all non-recovery animals from each test and control group at the end of the study and on all recovery group animals at the end of the treatment-free period
- Following parameters were examined:
+ Urea Inorganic
+ phosphorus (P)
+ Glucose Aspartate aminotransferase (ASAT)
+ Total protein (Tot.Prot.)
+ Alanine aminotransferase (ALAT)
+ Albumin Alkaline phosphatase (AP)
+ Albumin/Globulin (A/G) ratio (by calculation)
+ Creatinine (Creat)
+ Sodium (Na+)
+ Total cholesterol (Chol)
+ Potassium (K+)
+Total bilirubin (Bili)
+ Chloride (Cl-)
+ Bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: urinalytical investigations were performed on all non-recovery test and control group animals during week 12 and on all recovery group animals during the final week of the recovery phase.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Following parameters were examined:
+ Volume
+ Ketones
+ Specific Gravity
+ Bilirubin
+ pH
+ Urobilinogen
+ Protein Blood
+ Glucose Appearance

NEUROBEHAVIOURAL EXAMINATION: Yes : See detailed clinical observation


OTHER:Oestrus Cycle
During Weeks 12 and 13 a vaginal smear was taken daily and a sample was placed on a glass slide for all non-recovery group animals. The same procedure was undertaken for all recovery group females during Weeks 16 and 17. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrus was recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
+ All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
+ Organ Weights
- Adrenals
- Ovaries
- Brain
- Spleen
- Epididymides
- Testes
- Heart
- Thymus
- Kidneys
- Uterus (with cervix)
- Liver

HISTOPATHOLOGY: Yes
-Adrenals
- Pancreas
-Aorta (thoracic)
- Pituitary
- Bone & bone marrow (femur including stifle joint) •
- Prostate
- Bone & bone marrow (sternum)
- Rectum
- Brain (including cerebrum, cerebellum and pons)
- Salivary glands (submaxillary)
- Caecum
- Sciatic nerve
- Colon Seminal vesicles (including coagulating gland)
- Duodenum
- Epididymides ♦
- Skin (hind limb)
- Eyes* (including retina and optic nerve)
- Spinal cord (cervical, mid-thoracic and lumbar)
- Gross lesions
- Heart
- Spleen
- Ileum (including Peyer’s patches)
- Stomach
- Jejunum
- Testes ♦
- Kidneys
- Thymus
- Liver
- Thyroid/Parathyroid
- Lungs (with bronchi) #
- Tongue•
- Lymph nodes (mandibular and mesenteric)
- Trachea
- Mammary glands
- Urinary bladder
- Muscle (skeletal)•
- Uterus (with cervix)
- Oesophagus
- Vagina
- Ovaries

• These tissues were retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Bouin’s fluid and then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Detailed qualitative examination of the testes were undertaken on all non-recovery control and high group males, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatmentrelated effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related liver changes, examination was subsequently extended to include similarly prepared sections of liver from animals in the remaining dose groups

Other examinations:

Sperm Assessment

At necropsy the following procedures were performed on all non-recovery and recovery males:

i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility using an automated semen analyser to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) A sample of semen was preserved in formalin and then stained with eosin. A subsample was placed on a glass slide with a coverslip and a morphometric analysis of sampled semen was performed manually. A minimum of 200 individual sperm were assessed (wherever possible).
v) The cauda epididymis was separated from the body of the epididymis and then weighed. The cauda epididymis was then stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.

Oestrous cycle assessments

Statistics:

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 ProvantisTM 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 covarities. 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 Dunnett’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 (nonparametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
In addition, histopathological findings were analysed using Fisher’s exact test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See table 7.5.1/1

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no unscheduled deaths. There were no clinically observable signs of toxicity.

BODY WEIGHT AND WEIGHT GAIN:
No adverse effect on body weight change was detected on the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
No adverse effect on dietary intake was detected.

FOOD EFFICIENCY:
Food efficiency was unaffected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.

OPHTHALMOSCOPIC EXAMINATION:
No treatment-related ocular effects were detected for treated animals prior to termination of treatment.

HAEMATOLOGY:
No toxicologically significant effects were detected

CLINICAL CHEMISTRY:
Males treated with 39.2 mg/kg bw/day AI showed statistically significant increases in alanine aminotransferase and aspartate aminotransferase levels compared to controls, with a majority of values outside of the normally expected ranges.

Females treated with 39.2 mg/kg bw/day AI showed a statistically significant reduction in total protein, albumin and calcium levels, with the reductions in albumin and calciumextending into the 13.1 mg/kg bw/day AI dose group. Calcium values were also lower at the lowest treatment level when compared to controls. A number of the individual values were lower than the expected ranges. Males treated at all dose levels also showed a reduction in total protein and calcium levels. Although a convincing dose-related response was not evident for males, some values were outside of the normally expected range.

Males treated with 39.2 mg/kg bw/day AI also showed a statistically significant increase in inorganic phosphorus levels when compared to controls, with seven out of ten values outside of the normally expected ranges. Recovery high dose males showed a statistically significant reduction in inorganic phosphorus levels when compared to controls, with five values lower than the expected ranges. Although the aetiology of these intergroup differences is not clear, the contradictory values would suggest that these differences were of minimal toxicological significance.

Recovery high dose males showed an increase in glucose levels, when compared to their concurrent control values. No such effects were detected for non-recovery high dose males after the ninety day treatment period, and as such, this increase was considered to have arisen incidentally.

Recovery high dose females showed a statistically significant increase in chloride levels when compared to controls. All individual values were within the normally expected ranges, and the increase was considered to have been attributable to one lower than expected control value, and was therefore considered to be unrelated to treatment.

URINALYSIS: There were no treatment-related effects detected in the urinlytical parameters measured. Statistical analysis of the quantitative data not reveal any significant intergroup differences.

NEUROBEHAVIOUR:
There were no treatment-related effects

ORGAN WEIGHTS: There were no toxicologically significant organ weight changes detected.

GROSS PATHOLOGY:
Mottled appearance and pallor of the liver was observed at 39.2 mg/kg bw/day AI. No further treatment-related macroscopic abnormalities were detected.

HISTOPATHOLOGY (see Table 7.5.1/1):
LIVER: Hepatocellular eosinophilic cytoplasmic vacuoles were observed in animals of either sex treated with 13.1 and 39.2 mg/kg bw/day AI, and males treated with 6.54 mg/kg bw/day AI. Among these animals, hepatocellular single cell necrosis and
microvesicular hepatocellular degeneration were observed in males treated with 13.1 mg/kg bw/day AI and animals of either sex treated with 39.2 mg/kg bw/day AI. In the recovery animals, hepatocellular eosinophilic cytoplasmic vacuoles were still present for animals of either sex treated with 39.2 mg/kg bw/day AI. Increased incidence and/or severity of inflammatory cell foci were recorded in animals of either sex treated with 39.2 mg/kg bw/day AI. This was not evident in the recovery high dose animals.
One male and three females treated with the high dose level showed minimal periportal hypertrophy observed in hepatocytes not containing eosinophilic vacuole, and minimal bile duct proliferation was recorded in three males and two females treated with 39.2 mg/kg bw/day AI. This was not evident in the recovery high dose animals.

OTHER FINDINGS:
- Sperm Analysis: There were no toxicologically significant effects detected in sperm motility values, morphological assessments or in homogenisation-resistant spermatid counts. No treatment-related changes were detected for sperm morphology, homogenisation resistance and sperm motility. Statistical analysis of the data did not reveal any statistically significant intergroup differences.
-Oestrous cycle assessments: no treatment-related effects were detected.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

6.54 mg/kg bw/ day

Sex:

male

Basis for effect level:

other: based on the blood chemical changes and microscopic changes detected in the liver

2

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

13.1 mg/kg bw/day

Sex:

female

Basis for effect level:

other: based on the blood chemical changes and microscopic changes detected in the liver

3

Dose descriptor:

LOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

13.1 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Based on the microscopic changes observed in the liver

4

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

39.2 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Based on the microscopic changes observed in the liver

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

Table 7.5.1/1: Incidence and mean severity grade of main findings in Liver

 

Finding Incidence/ MeanSeverityGrade	Group1		Group2		Group3		Group4
Non-recovery	10M	10F	10M	10F	10M	10F	10M	10F
Hepatocellulareosinophiliccytoplasmicvacuole,periportalIncidence/MeanSeverity	0	0	2/1.0	0	9/1.3**	1/1.0	10/2.2**	10/1.6**
Microvesicularhepatocellulardegeneration,periportalIncidence/MeanSeverity	0	0	0	0	1/1.0	0	4/2.3*	1/1.0
Singlecellnecrosis,hepatocellular,mainlyperiportalIncidence/MeanSeverity	0	0	0	0	3/1.0	0	10/1.9**	10/1.3**
Inflammatorycellfoci Incidence/MeanSeverity	5/1.0	3/1.0	6/1.0	4/1.0	9/1.1	4/1.0	10/2.0*	10/1.3**
Bileductproliferation Incidence/MeanSeverity	0	0	0	0	0	0	3/1.0	2/1.0
Hepatocellularhypertrophy,periportalIncidence/MeanSeverity	0	0	0	0	0	0	1/1.0	3/1.0
Recovery	10M	10F					10M	10F
Hepatocellulareosinophiliccytoplasmicvacuoles,periportalIncidence/MeanSeverity	0	0					10/1.0**	7/1.0**
Inflammatorycellfoci Incidence/MeanSeverity	6/1.0	5/1.0					9/1.0	4/1.0

Fisher’s Exact Test (one-sided): Statistically different from Group 1 (*, p < 0.05; **, p < 0.01)

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, the administration of Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea by oral gavage for a period of ninety consecutive days resulted in treatment-related effects at all dose levels.
The effects were confined to blood chemical changes and microscopic changes detected in the liver. Based on the results of this study, a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 6.54 mg/kg bw/day active ingredient for males and 13.1 mg/kg bw/day active ingredient for females.

Executive summary:

In a sub-chronic study performed according to OECD 408 guideline and in compliance with GLP (Harlan, 2013), Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea (THPC-urea) was administered continuously by gavage to three groups, each of ten male and ten female Wistar rats, for 90 consecutive days, at dose levels of 6.54, 13.1 and 39.2 mg/kg bw/day (incorporating a correction factor for 65.4% purity). A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Two recovery groups, each of ten males and ten females, were treated with the high dose (39.2 mg/kg bw/day AI) or the vehicle alone for 90 consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake, water consumption and oestrus cycles were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all animals at the end of the treatment and recovery period. Ophthalmoscopic examination was also performed on non-recovery control group and high dose animals. Sperm analysis was also performed at the end of the treatment and recovery periods. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

No treatment-related effects were detected on Behavioural Assessment, Functional Performance Tests, Sensory Reactivity Assessments, Body Weight, Food Consumption, Oestrous Cycle Assessments, Ophthalmoscopy, Haematology, Urinalysis, Organ Weights and sperm analysis. However, the males treated with 39.2 mg/kg bw/day AI showed increases in alanine aminotransferase and aspartate aminotransferase levels compared to controls. Females treated with 39.2 mg/kg bw/day AI showed a reduction in total protein, albumin and calcium levels, with the reductions in albumin and calcium extending into the 13.1 mg/kg bw/day AI dose group. Calcium values were also lower at the lowest treatment level. Males treated at all dose levels also showed a reduction in total protein and calcium levels.

At necropsy, mottled appearance and pallor of the liver was recorded for three males treated at 39.2 mg/kg bw/day AI. One female treated with 39.2 mg/kg bw/day AI also showed a mottled appearance of the liver. No further treatment-related macroscopic abnormalities were detected.

Histopathological findings show treatment-related changes in the liver. Hepatocellular eosinophilic cytoplasmic vacuoles were observed in animals of either sex treated with 13.1 and 39.2 mg/kg bw/day AI, and males treated with 6.54 mg/kg bw/day AI. Among these animals, hepatocellular single cell necrosis and microvesicular hepatocellular degeneration were observed in males treated with 13.1 mg/kg bw/day AI and animals of either sex treated with 39.2 mg/kg bw/day AI. In the recovery animals, hepatocellular eosinophilic cytoplasmic vacuoles were still present for animals of either sex treated with 39.2 mg/kg bw/day AI. Increased incidence and/or severity of inflammatory cell foci were recorded in animals of either sex treated with 39.2 mg/kg bw/day AI. This was not evident in the recovery high dose animals. One male and three females treated with the high dose level showed minimal periportal hypertrophy observed in hepatocytes not containing eosinophilic vacuole, and minimal bile duct proliferation was recorded in three males and two females treated with 39.2 mg/kg bw/day AI. This was not evident in the recovery high dose animals.

Under the test conditions of this study, the administration of Tetrakis (hydroxymethyl) phosphonium chloride, oligomeric reaction products with urea by oral gavage for a period of ninety consecutive days resulted in treatment-related effects at all dose levels. The effects were confined to blood chemical changes and microscopic changes detected in the liver. Based on the results of this study, a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 6.54 mg/kg bw/day active ingredient for males and 13.1 mg/kg bw/day active ingredient for females.