Phosphoric acid, mono- and di-C16-18-alkyl esters

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 August 2017 to XXXX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

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:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: See remarks

Version / remarks:

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(octadecyloxy)phosphonic acid

EC Number:

939-526-9

Cas Number:

90506-73-3

Molecular formula:

C18H39O4P

IUPAC Name:

(octadecyloxy)phosphonic acid

Test material form:

other: solid

Details on test material:

Description: phosphoric acid, octadecyl ester
Batch Number: CEB5-90-3
Purity: 100%
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%, UVCB substance
Storage Conditions: Room temperature in the

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 195 to 229g, the females weighed 131 to 176g, and were approximately seven weeks old.

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., UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 +/- 3 deg.C and 50 +/- 20% respectively.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

arachis oil

Details on oral exposure:

The test material was heated to approximately 85 deg.C to form a solution in Arachis oil at 6 mL/kg and to ensure the test material formulations remained in a dosable state for the duration of the dosing period. During the dosing procedure the test material formulations and control vehicle were kept in a warming bath at approximately 46 deg.C.

The volume of test and control material administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test material formulations were taken and analyzed for concentration of test material at Envigo Research Limited, Shardlow, UK, Analytical Services.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Treatment was suspended for animal welfare reasons at the highest treatment group on Days 13/12 (males and females, respectively). Treatment recommenced approximately 48 hours later at a reduced dose level of 500 mg/kg/bw/day.

No. of animals per sex per dose:

Ten males and ten females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill. Additional unscheduled body weights were performed for each dose group for the monitoring of animal health.

Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behavior
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin color
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988).

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 occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and 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. 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).

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 scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:

Grasp response
Vocalization
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

The eyes of all Groups 1 to 4 animals were examined pre-treatment. During Week 12, the eyes of all control and high dose animals (Groups 1 and 4, respectively) were examined. Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90); see Deviations from Study Plan. Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

Hematology:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)

Total leukocyte count (WBC)
Differential leukocyte 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’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)

Sacrifice and pathology:

On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ weights:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Uterus

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

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
¿ Preserved in Modified Davidson’s fluid

All tissues were dispatched to the Test Site for processing. All tissues from control and 1000/500 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 micro m and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the adrenal glands from animals in the low and intermediate groups.

Pathology:
Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology results for the study was conducted by an appointed Responsible Scientist working at a designated Test Site.

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, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed 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 analyzed 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 (non-parametric).

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000/500 mg/kg bw/day, all surviving animals showed sporadic but occasionally repeated incidences of noisy respiration throughout the study. Instances of noisy respiration, with an concomitant increased incidence in increased post-dosing salivation, are frequently observed when test material formulations of an unpalatable or irritant nature are dosed via the oral gavage route. However, whilst the majority of surviving males showed occasional incidences of post-dosing salivation, this was only apparent for one surviving female, possibly indicating that the finding was due more to the physical properties of the test material formulation rather than irritancy. Two surviving males also had incidences of piloerection, decreased respiration rate and lethargy, with one also exhibiting hunched posture during the study. The similarity of these signs to those seen in the decedent animal suggests a similar underlying cause; solidification of the formulations in the gastro-intestinal tract impacting on normal gastro-intestinal function.

At 300 mg/kg bw/day, all surviving animals exhibited sporadic but occasionally repeated incidences of noisy respiration throughout the study, but only one male exhibited instances of increased post-dosing salivation. One further male had an isolated incidence of labored respiration and decreased respiration rate.

At 100 mg/kg bw/day, six males and three females showed sporadic incidences of noisy respiration throughout the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were ten premature deaths during the study: one control female, two males at 300 mg/kg bw/day, and three males and four females at 1000 mg/kg bw/day.

At 1000 mg/kg bw/day, four females were killed in extremis on Day 12 after exhibiting body weight losses of 28 to 30 grams between Days 8-12 and adverse clinical signs, which included distended abdomen, hunched posture, hypothermia, pilo-erection, gasping, labored and noisy respiration, deceased respiratory rate, and lethargy. Macroscopic examination at necropsy revealed white colored contents (considered to be test material) in the stomach, gaseous distension in the stomach, jejunum, duodenum, ileum, and cecum. Two of these females were also observed to have a small spleen.

Two males receiving 1000 mg/kg bw/day were killed in extremis on Day 13 after exhibiting body weight losses of 38 grams between Days 8-13 and adverse clinical signs, which included hypothermia, pallor of the extremities, lethargy, and noisy and gasping respiration. One further animal treated at 1000/500 mg/kg bw/day was killed in extremis on Day 71 after exhibiting similar adverse clinical signs. Macroscopic examination of these males at necropsy revealed white colored contents (considered to be test material) in the stomach, gaseous distension in the jejunum, duodenum, ileum, and cecum. Two of the males had small spleens and two were observed with small seminal vesicles, one of which also had small epididymides.

It is considered the deaths of these animals at 1000 mg/kg bw/day were most likely associated with the physical nature of the unabsorbed test material rather than reflecting true systemic toxicity. At room temperature, the formulation becomes a white wax like substance, and the formulations administered to the animals were warmed to keep them in a liquid state suitable for dosing. However, there was no way of preventing solidification of the formulation once it has reached the stomach and this process probably impacted on normal gastro-intestinal function, leading to a decline in physical condition for some of the animals.

One male receiving 300 mg/kg bw/day was killed in extremis on Day 77, due to a body weight loss of 38g between Days 71-77, and adverse clinical signs including hunched posture, lethargy, pilo-erection, gasping and noisy respiration, and deceased respiratory rate. Macroscopic examination at necropsy revealed white colored contents (considered to be test material) in the stomach and duodenum and gaseous distension in the jejunum, duodenum and ileum. In view of the similarity of necropsy findings to those of decedents at 1000 mg/kg bw/day, this death is similarly considered to reflect the intrinsic physical properties of the test material. A second male at 300 mg/kg bw/day was found dead on Day 3 of treatment; no clinical signs had been apparent for this animal prior to death. Necropsy revealed dark lungs, which is not unusual for animals found dead, and microscopic examination of issues did not reveal the cause of death. This death occurred earlier than the deaths at the high dose. While the aetiology of this death was not established, in the absence of deaths due to systemic toxicity at 1000 mg/kg bw/day, this death at 300 mg/kg bw/day was considered most likely to be incidental and unrelated to treatment.

One control female was found dead and cannibalised on Day 21 of treatment; no clinical signs had been apparent for this animal prior to death. Although, there were no significant findings noted upon macroscopic or microscopic examination, the extent of cannibalization precluded any meaningful assessment; the aetiology of this incidental death could not be established.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, animals of either sex had additional body weights performed on Day 13/12 of treatment (males/females, respectively) to assess animal health, due to body weight losses being noted in the animals which were killed in extremis. Additional daily body weight measurement continued for all dose groups from Day 14/13 (males/females, respectively) until Day 22 to assess animal health and body weight development.

At 1000/500 mg/kg bw/day, mean body weight gains of males were generally similar to control throughout the study. Whilst the values for mean body weight gain appeared to contradict the body weight losses noted for the decedent animals, similar sporadic body weight losses were apparent for five of the surviving animals at this dosage. Lower mean bodyweight gains did attain statistical significance compared to controls during Week 8 (Days 50-57) and Week 13 (85-91), and a mean body weight loss was recorded for Week 10 (Days 64-71); on all occasions one or more males showed a body weight loss. No similar episodes of body weight losses were observed for control males. Overall bodyweight gain of surviving males from the start of treatment was slightly lower than control at termination and, when overall gain was measured from Day 15 of the study (following the reduction of the dosage to 500 mg/kg bw/day), differences from control attained statistical significance, but were regarded as insufficient to represent an adverse effect.

For females at 1000/500 mg/kg bw/day, there were no statistically significant differences in body weight gain apparent throughout the study. Overall body weight measured from the start of treatment and from Day 15 of the study was slightly lower than control at termination, but differences showed no statistical significance. Although sporadic occasions of body weight loss were noted for individual females from Day 43 of the study, these were consistent with the normal body weight fluctuations expected as the weight of the females plateau with age, and the incidence of these weight losses was similar to control.

There were no statistically significant differences from control for mean body weight gain for either sex at 100 or 300 mg/kg bw/day. Three surviving males at 300 mg/kg bw/day each showed a single body weight loss during the study; one of these body weight losses that was apparent at Week 4 was more than fully resolved the following week, and the other two losses occurred towards the end of the study. For females at 100 or 300 mg/kg bw/day, sporadic occasions of body weight loss were noted for individual females, mainly from Day 43 of the study; the incidence of these weight losses was similar to control and they were considered to be consistent with the normal body weight development for this sex.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no obvious adverse effects on food consumption for animals of either sex at 100, 300 or 1000/500 mg/kg bw/day. Food consumption for treated animals tended to be slightly lower than control, but the lack of any consistent dosage relationship suggest that these differences reflected normal biological variation rather than an effect of treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

Intergroup differences in food conversion efficiency did not indicate any obvious effect of treatment for surviving animals at 100, 300 or 1000/500 mg/kg bw/day.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of animals of both sexes from the control and surviving 1000/500 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects on the hematological parameters measured for either sex at 100, 300 or 1000/500 mg/kg bw/day.

For females at 300 and at 1000/500 mg/kg bw/day, mean total leukocyte count was statistically significantly higher than control (p<0.01) due to statistically significantly higher mean numbers of lymphocytes (p<0.01) and neutrophils (p<0.01 and p<0.05 respectively). At 1000/500 mg/kg bw/day, mean eosinophil counts were also statistically significantly higher than control (p<0.05). An increase in white cells, particularly lymphocytes and neutrophils, may be associated with an inflammatory response to test material exposure, although specific histopathology associated with inflammatory responses was not observed. The majority of individual values for these parameters were within the respective historical control ranges and, in the absence of any supporting histopathological correlates, these finding were considered to be of no toxicological significance.

For males at 300 and at 1000/500 mg/kg bw/day, mean neutrophil counts were as statistically significantly higher than control (p<0.05) but there was no accompanying significant increase in mean total leukocyte count. The majority of individual values for treated males were within the historical control range and, in isolation and in the absence of any supporting histopathological correlates, this finding were considered to be of no toxicological significance.

Males at 1000/500 mg/kg bw/day showed a slightly shorter activated partial thromboplastin time (p<0.01) and a longer prothrombin time (p<0.05) compared to controls. However all individual values were within the background control ranges and, in the absence of any histopathological correlates, these findings were considered to be of no toxicological significance.

For females at all dosages, statistically significant higher mean hemoglobin (p<0.05), hematocrit (p<0.05) and total erythrocytes counts (p<0.05 – p<0.01) were apparent compared to control, but values showed no dosage-relationship. Whilst the majority of individual values for treated females were within the respective historical control ranges, the majority of control values were below these historical ranges. These findings, in the absence of any histopathological correlates, were considered to reflect low control values and were therefore incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects on the blood chemistrycal parameters measured for either sex at 100, 300 or 1000/500 mg/kg bw/day.

For females at all doses, higher mean calcium concentrations attained statistical significance (p<0.05 – p<0.01); when compared to control; although the highest mean value was at 1000/500 mg/kg bw/day, mean values at lower doses showed no dose-response. All individual values for treated females were within the historical control range and, without any histopathological correlates, this findings were considered to be of no toxicological significance.

For females at all doses, lower mean chloride concentrations attained statistical significance (p<0.05) when compared to control, but there was no dose-response. All individual values for treated females were within the historical control range and, in the absence of any histopathological correlates, theseis findings were considered to be of no toxicological significance.

For females at 1000/500 mg/kg bw/day, higher creatinine levels concentrations attained statistical significance (p<0.05) when compared to control. All individual values were within the background control ranges and, without any histopathological correlates, these findings were considered to be of no toxicological significance.

There were no statistically significance differences from control apparent for males at 100, 300 or 1000/500 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Behavioral Assessments
Observations during behavioral assessments were consistent with the clinical signs that had been apparent throughout the study and were considered not to indicate any underlying neurological effect of treatment.

Instances of noisy respiration were apparent at 300 and 1000/500 mg/kg bw/day, and to a lesser extent at 100 mg/kg bw/day. At 1000/500, two males had isolated incidences of slight piloerection, with one male also showing decreased respiration rate.

At 300 mg/kg bw/day one male showed respiratory distress and slight pilo-erection on Day 77 and was subsequently killed in extremis (see mortalities).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000/500 mg/kg bw/day males showed a statistically significant (p<0.05) increase in kidney weight both absolute and relative to terminal body weights, a true dose related response was not apparent. All individual values were within the background control ranges and in the absence of any histopathological correlates this finding was considered to be of no toxicological significance.

No such effects were detected in 1000/500 mg/kg bw/day females or animals of either sex at 100 or 300 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For surviving animals, neither the type, incidence or distribution of macroscopic findings apparent at terminal necropsy indicated any effect of treatment. In particular, the accumulation of test material in the gastro-intestinal tract observed for many decedent animals on the study was not apparent at terminal necropsy.

Red discoloration of the lungs was observed for one control female, one male and three females receiving 100 mg/kg bw/day, one male and two females receiving 300 mg/kg bw/day, and one female receiving 1000 mg/kg bw/day. This finding is frequently observed with the method of euthanasia employed in this study and was considered incidental and unrelated to treatment.

The remaining isolated findings observed for animals at terminal necropsy were considered to be incidental and unrelated to treatment.

Adrenal Glands
Hypertrophy of the zona glomerulosa was increased in males from all treated groups. It was present in three, five and all males treated with 100, 300 and 1000/500 mg/kg bw/day, respectively. In females, this was variable and did not show a clear response to the test material. The zona glomerulosa is the site of synthesis of aldosterone a mineralocorticoid, which is mainly involved in the control of salt and water balance in the body. Secretion of aldosterone is controlled through the rennin-angiotensin system and hypertrophy of the zona glomerulosa is generally considered to be an adaptive process following stimulation of this system (Domenici Lombardo, 1990; Greaves 2007).

No other changes were noted which could be attributed to treatment with the test material at terminal kill.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Premature Decedents
There were ten early deaths on the study in total.

One male at dosed at 300 mg/kg bw/day and one control female, found dead on Days 3 and 21 of treatment, respectively, showed no clinical signs prior to death. The majority of tissues from the control female were cannibalized, and there were no significant findings present at the macroscopic or microscopic examination for either animal.

Two males and four females in the 1000 mg/kg bw/day group that were killed in extremis on Days 12 or 13 showed non-specific evidence of stress or poor condition, with thymic atrophy and depletion/atrophy of other tissues. All had minor hyperplasia of the tracheal epithelium. A cause of death could not be determined, but is considered likely to be related to the formulation of the test material.

A third male dosed at 1000 mg/kg bw/day was killed in extremis on Day 71. Histopathology revealed non-specific evidence of stress or poor condition, with thymic atrophy and depletion/atrophy of other tissues. There were also minor inflammatory changes in the caecum and rectum, and zona glomerulosa hypertrophy in the adrenal glands.

One male dosed at 300 mg/kg bw/day and killed in extremis on Day 77 exhibited hypertrophy of the zona glomerulosa in the adrenal glands and splenic depletion. The cause of death was not determined.

Administration of the test material at 1000 mg/kg bw/day resulted in the death of 6/20 animals on Days 12 and 13, potentially related to the formulation of the test material. A further death at this dosage occurred on Day 71. The death of one animal administered the test material at 300 mg/kg bw/day in the final month of the study is considered unlikely to be of toxicological significance.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in the functional parameters measured.

Females treated with 100 mg/kg bw/day had a statistically significant reduction (p<0.01) in hindlimb grip strength. The intergroup difference was confined to one out of the three tests and, in the absence of any similar effects at higher doses, this finding was considered to be incidental and of no toxicological importance.

There were no toxicological changes in the sensory parameters measured.

Details on results:

Assessment of toxicity during this study was made more difficult by the physical nature of the test material formulations. Formulations were prepared in Arachis oil and kept warm prior to and during the dosing procedure, which ensured that the formulations remained in a liquid state. At room temperature the formulation solidifies to form a white waxy-like solid. Whilst every effort was made to ensure formulations were acceptable for dosing, the subsequent solidification of the test material formulation within the gastro-intestinal tract was an inherent risk with this particular material.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 500 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

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the No Observed Adverse Effect level (NOAEL) for systemic toxicity when administered daily over ninety consecutive days was considered to be 500 mg/kg bw/day.
Deaths occurred at 1,000 mg/kg bw/day, which were considered to result from the intrinsic physical properties of unabsorbed, solidified test material in formulation rather than any systemic effect of treatment. Due to this mortality at 1,000 mg/kg bw/day, the highest dose was reduced to 500 mg/kg bw/day. At this dosage, overall male body weight gain was slightly lower than control, but at the level observed was regarded as insufficient to represent an adverse effect. Hypertrophy of the zona glomerulosa of the adrenal glands for males was also apparent at this dosage, but was considered to be adaptive in nature and not adverse. The establishment of the maximum NOAEL for systemic toxicity is hampered by the inherent physical properties of the test material and may be higher than the 500 mg/kg bw/day dose level achieved in this study.

Executive summary:

This study was designed to investigate the systemic toxicity of the test material.

The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 100, 300 and 1000 mg/kg bw/day. Treatment was suspended for the highest treatment group on Days 13/12 (males and females, respectively). Treatment recommenced approximately 48 hours later at a reduced dose level of 500 mg/kg/bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on all study animals before the start of treatment and on control and high dose animals during Week 12 of treatment.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all control and high dose animals (Groups 1 and 4) as well as any gross lesions from Groups 1 to 4 was performed in the first instance. As there were treatment-related findings in the adrenal glands, examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups.

There were ten premature deaths during the study: one control female, two males at 300 mg/kg bw/day, and three males and four females at 1000 mg/kg bw/day. Seven animals dosed with 1000 mg/kg bw/day were killed in extremison Day 12or 13of treatment after exhibiting significant body weight losses and adverse clinical signs including distended abdomen, hunched posture, hypothermia, lethargy, pilo-erection, gasping, labored and noisy respiration, and deceased respiratory rate, and lethargy. These animals exhibited similar macroscopic findings,including white colored contents in the stomach,  gaseous distension throughout the gastrointestinal tract, and small spleen (two animals per sex).  Two males were observed with small seminal vesicles, one of which also had small epididymides.

 

The deaths of these animals at 1000 mg/kg bw/day were considered most likely associated with the inherent physical nature of the test material rather than reflecting true systemic toxicity. At room temperature, the formulation becomes a white wax-like substance, and the formulations administered to the animals were warmed to keep them in a liquid state suitable for dosing. However, there was no way of preventing solidification of the formulation once it reached the stomach and resolidification probably impacted normal gastro-intestinal function, leading to a decline in physical condition for some of the animals.

 

One male receiving 300 mg/kg bw/day was killed in extremis on Day 77 due to a body weight loss of 38g between Days 71-77, and adverse clinical signs including hunched posture, lethargy, pilo-erection, gasping and noisy respiration, and deceased respiratory rate.  Macroscopic examination at necropsy revealed findings in the gastrointestinal tract similar to those observed among animals receiving 1000 mg/kg bw/day that were killed in extremis, including white colored contents (considered to be test material) in the stomach and duodenum.  This death was considered to reflect the intrinsic physical properties of unabsorbed test material.  

 

The deaths of two animals found dead were unexplained.  One male at 300 mg/kg bw/daywas found dead on Day 3; no clinical signs had been apparent for this animal prior to death and necropsy only revealed dark lungs. While the aetiology of this death was not established, this death was considered most likely to be incidental and unrelated to treatment.  One control female was found dead and cannibalised on Day 21 of treatment; no clinical signs had been apparent prior to death and the extent of cannibalization precluded any meaningful macroscopic or microscopic assessment. The aetiology of this incidental death could not be established.

Clinical Observations

At 1000/500 mg/kg bw/day, all surviving animals showed sporadic but occasionally repeated incidences of noisy respiration throughout the study. Occasional incidences of increased post-dosing salivation were observed for the majority of surviving males and one surviving female. 

Two surviving males also showed incidences of piloerection, decreased respiration rate and lethargy, with one also showing hunched posture during the study. 

At 300 mg/kg bw/day, all surviving animals showed sporadic but occasionally repeated incidences of noisy respiration throughout the study. One male showed instances of increased post-dosing salivation and one further male showed an isolated incidence of labored respiration and decreased respiration rate.       

At 100 mg/kg bw/day, six males and three females showed sporadic incidences of noisy respiration throughout the study. 

Behavioral Assessment

Instances of noisy respiration were apparent at 300 and 1000/500 mg/kg bw/day, and to a lesser extent at 100 mg/kg bw/day. At 1000/500, two males showed isolated incidences of slight piloerection, with one male also showing decreased respiration rate. These observations s were consistent with the clinical signs apparent throughout the study and were considered not to indicate any underlying neurological effect of treatment. 

At 300 mg/kg bw/day one male (No. 46) showed respiratory distress and slight pilo-erection on Day 77 and was subsequently killedin extremis(see mortalities).

Functional Performance Tests

There were no toxicologically significant changes in the functional parameters measured at 100, 300 and 1000/500 mg/kg bw/day.

Sensory Reactivity Assessments

There were no toxicological changes in the sensory parameters measured at 100, 300 and 1000/500 mg/kg bw/day.

Body Weight

There were no adverse effects of treatment in body weight gains from surviving animals at 100, 300 and 1000/500 mg/kg bw/day.

At 1000/500 mg/kg bw/day, sporadic body weight losses were apparent for five of the surviving animals and overall bodyweight gain of surviving males was slightly lower than control at termination. Three surviving males at 300 mg/kg bw/day also showed a single episode of body weight loss during the study.

Food Consumption

There were no adverse effect on food consumption or food conversion efficiency for animals of either sex at 100, 300 or 1000/500 mg/kg bw/day.

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Ophthalmoscopy

There were no treatment related ocular effects detected for control or high dose animals.

Hematology

Assessment of hematology parameters did not indicate any toxicologically significant effects of treatment for either sex at 100, 300 or 1000/500 mg/kg bw/day.

Blood Chemistry

Assessment of blood chemistry parameters did not indicate any toxicologically significant effects of treatment for either sex at 100, 300 or 1000/500 mg/kg bw/day.

Necropsy

There were no toxicologically significant abnormalities detected for surviving animals at 100, 300 or 1000/500 mg/kg bw/day.

Organ Weights

No toxicologically significant effects were detected in the organ weights measured in animals of either sex at 100, 300 or 1000/500 mg/kg bw/day.

Histopathology

Premature Decedents

There were ten early deaths on the study.

One 300 mg/kg bw/day male (Day 3) and one control female (Day 21) were found dead but showed no significant findings at macroscopic or microscopic examination. The majority of tissues from the control female were cannibalized.

The following animals treated with 1000 mg/kg bw/day were killed in extremison days 12 or 13: 

Males 64 and 70; and Females 72, 74, 78 and 80 at histopathology had non-specific evidence of stress or poor condition with thymic atrophy and depletion/atrophy of other tissues. All had minor hyperplasia of the tracheal epithelium. A cause of deterioration could not be determined but is considered likely to be related to the test material formulation.

Male 61, treated with 1000/500 mg/kg bw/day and killed in extremison Day 71, showed gaseous distention of the gastrointestinal tract. Histopathology showed non-specific evidence of stress or poor condition with thymic atrophy and depletion/atrophy of other tissues. There were also minor inflammatory changes in the caecum and rectum. Zona glomerulosa hypertrophy in the adrenal glands was also observed.

Male 46, treated with 300 mg/kg bw/day, was killed in extremison Day 77 with similar clinical and macroscopic changes as those exhibited by the 1000 mg/kg bw/day decedents. It had hypertrophy of the zona glomerulosa in the adrenal glands; splenic depletion was also noted. The cause of deterioration was not determined.

Terminal Kill

Adrenal Glands 

There was an increase in hypertrophy of the zona glomerulosa in males from all treated groups. Mild hypertrophy was present in three, five and all males at 100, 300 and 1000/500 mg/kg bw/day respectively.  In females, this change was variable and did not show a clear response to the test material. This is generally considered to be an adaptive change which may be stress related.

No other changes were noted which could be attributed to treatment with the test material at terminal kill.

Conclusion

Based on the results of this study, the No Observed Adverse Effect level (NOAEL) for systemic toxicity when administered daily over ninety consecutive days was considered to be 500 mg/kg bw/day. Although a number of deaths occurred on the study at this dosage, these deaths were considered to result from the intrinsic physical properties of unabsorbed test material formulation rather than any systemic effect of treatment. At this dosage, overall male body weight gain was slightly lower than control, but the level observed was regarded as insufficient to represent an adverse effect. Hypertrophy of the zona glomerulosa of the adrenal glands for males was also apparent at this dosage, but was considered to be adaptive in nature and not adverse. The establishment of the maximum NOAEL for systemic toxicity is hampered by the inherent physical properties of the test material and may be higher than the 500 mg/kg bw/day dose level achieved in this study.