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Description of key information

The purpose of this study was to assess the neurotoxic effects and systemic toxic potential of tBuTPP Low TPP (an industrial chemical)

when administered orally, by gavage, to Han Wistar rats for 13 weeks.

Three groups of 16 male and 16 female RccHan™;WIST rats received tBuTPP Low TPP at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. A similarly constituted control group received the vehicle, corn oil, at the same volume dose and over the same treatment period as treated groups.

The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day in males and 1000 mg/kg/day in females. The NOAEL for neurotoxicity was 1000 mg/kg/day in both sexes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
As per ECHA Decision number: CCH-D-2114449848-30-01/F
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
revised 25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
EC number 939-505-4
Intended use Industrial chemical.
Appearance Clear, transparent, yellow liquid.
Storage conditions At ambient temperature (15 to 25°C) in the dark
Supplier Sponsor
Lot number 18235H0299
Expiry date 28 August 2022.
Purity Multi constituent substance, considered to be 100% when within specification.

Certificate of Ananlysis attached.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: RccHan:WIST rat.
Supplier: Envigo RMS Limited.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Number of animals 69 males and 69 females.
Acclimatization 12 days before commencement of treatment.
Age of animals at start of treatment 41 to 47 days.
Weight range of animals at the start of treatment Males: 128 to 171 g
Females: 107 to 150 g
Allocation Randomly allocated on arrival. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Animal Care and Husbandry
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24C and 40-70%. (Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.


Animal Accommodation
Cages were comprised of a polycarbonate body with a stainless steel mesh lid and were changed at appropriate intervals.
Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.

Number of animals per cage Up to four of the same sex.
Animals were separated into single housing overnight prior to functional observational battery testing. Following the completion of the functional observational battery testing the animals were returned to group housing.

Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment: Aspen gnawing material Provided to each cage throughout the study and replaced when necessary, except when animals were separated into single housing overnight prior to functional observational battery testing.

Plastic shelter was provided to each cage throughout the study and replaced when necessary, except when animals were separated into single housing overnight prior to functional observational battery testing.

Diet Supply
Diet Teklad 2014C, pelleted diet. The following batch numbers were used during this study: 082619MB; 010220MB and 030320MA.
Availability Non-restricted (removed overnight before blood sampling for hematology and blood chemistry.)

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Validation of the Analytical Method and Formulation Accuracy, Homogeneity and Stability in Corn Oil was done prior to performiance of the study.
Summary
The purpose of this study was to establish the procedures for the preparation and analysis of tBuTPP- low TPP in liquid formulations to support future toxicity studies. Validation was undertaken with respect to specificity of the chromatographic analysis, limit of detection, limit of quantification, linearity of detector response, precision of injection, calibration standard stability, extract stability, method accuracy and precision of nominal concentrations of 2.5 mg/mL and 250 mg/mL in corn oil. The homogeneity and stability were confirmed with respect to the level of concentration for tBuTPP- low TPP in corn oil formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL. Storage was confirmed at ambient temperature (15 to 25ºC) for up to 1 day and following refrigeration (2 to 8°C) for up to 15 days following fresh preparation.
Duration of treatment / exposure:
Target 90 days, actual a full 13 weeks.
Treatment continued until termination for each animal and serial observations were recorded at appropriate intervals
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
0 OB/kg bw/day (nominal)
Remarks:
Vehicle control group (corn oil) at the same volume dose and over the same treatment period as treated groups
No. of animals per sex per dose:
Three groups of 16 male and 16 female RccHan™;WIST rats
Control animals:
yes
yes, concurrent vehicle
Details on study design:
The purpose of this study was to assess the neurotoxic effects and systemic toxic potential of tBuTPP Low TPP (an industrial chemical) when administered
orally, by gavage, to Han Wistar rats for 13 weeks.

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan™;WIST strain was used because of the historical and positive control data available at this laboratory.

The oral route of administration was chosen to simulate the conditions of possible human exposure. The test item was administered by gavage.

Rationale for Dose Level Selection
The dose levels for this study were selected based on available toxicity data including the findings from a 28-Day Dose Range Finding Oral Toxicity Study in Wistar Rats with tBuTPP Low TPP conducted on behalf of the Sponsor by another Test Facility (BSL BIOSERVICE; BSL Munich Study No 192806). In that study, dose levels of 100, 300 or 1000 mg/kg/day were well-tolerated, with no adverse finding at any dose level. The same dose levels (100, 300 or 1000 mg/kg/day) and dose volume (4 mL/kg) were therefore selected for this longer-term study.



The purpose of this study was to assess the neurotoxic effects and systemic toxic potential of tBuTPP Low TPP (an industrial chemical) when administered orally, by gavage, to Han Wistar rats for 13 weeks.
Three groups of 16 male and 16 female RccHan™;WIST rats received tBuTPP Low TPP at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. A similarly constituted control group received the vehicle, corn oil, at the same volume dose and over the same treatment period as treated groups.
During the study, clinical condition, neurobehavioral screening, body weight, food consumption, water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, estrous cycle, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken
2.4 Rationale for Dose Level Selection
The dose levels for this study were selected based on available toxicity data including the findings from a 28-Day Dose Range Finding Oral Toxicity Study in Wistar Rats with tBuTPP Low TPP conducted on behalf of the Sponsor by another Test Facility (BSL BIOSERVICE; BSL Munich Study No 192806). In that study, dose levels of 100, 300 or 1000 mg/kg/day were well-tolerated, with no adverse finding at any dose level. The same dose levels (100, 300 or 1000 mg/kg/day) and dose volume (4 mL/kg) were therefore selected for this longer-term study.
Observations and examinations performed and frequency:
During the study, clinical condition, neurobehavioral screening, body weight, food consumption, water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, estrous cycle, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken.
Sacrifice and pathology:
During the study samples for organ weight, macropathology and histopathology investigations were undertaken.

Terminal Investigations
Method of Kill: Ten males and ten females per group with the lowest animal identity numbers (toxicity assessments) Carbon dioxide. Each animal was exsanguinated. Six males and six females per group with the highest animal identity numbers (neurotoxicity assessments). Overdose of barbiturate by intra-peritoneal injection. Heart exposed to permit perfusion with fixative via aorta.

Necropsy
In total, six males and six females per group (the animals bearing the highest identification numbers for each sex per group) were assigned to in situ perfusion for neurotoxicity assessments. The remaining ten males and ten females per group (the animals bearing the lowest identification numbers for each sex per group) were assigned to the standard necropsy procedures for 90-day toxicity assessments.
Animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Statistics:
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
Body weight, using gains over appropriate study periods
Food consumption, over appropriate study periods
Water consumption
Neurobehavioral data
Hematology
Blood chemistry
Organ weights, absolute and adjusted for terminal body weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4
Clinical signs:
no effects observed
Description (incidence and severity):
There were no unscheduled deaths and the clinical appearance of the animals was unaffected by treatment. There were no treatment-related findings at the in-cage, in-hand or arena observations or at the reactivity and motor activity investigations.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was persistently lower than control from Week 2 in males receiving 1000 mg/kg/day (overall 16% reduction, Week 0-13) with no corresponding decrease of food intake. In females, all animals receiving tBuTPP Low TPP gained more body weight than the controls during the first week of treatment, with the increased gain persisting throughout the study at 1000 mg/kg/day only (gain during Week 0-13 was 29% higher than control). This corresponded with a 21% overall increase of food intake in the animals.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males receiving 1000 mg/kg/day, water intake was 62 and 74% higher than control in males when quantified during Weeks 6 and 9, respectively and in females the increases were 74 and 77%, respectively. In males receiving 300 mg/kg/day, water intake was 21 and 29% higher than control in Weeks 6 and 9, respectively.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination after 13 weeks revealed the following treatment-related differences from control: high neutrophil count in both sexes receiving
300 or 1000 mg/kg/day (increases of 32 and 20% for males and 47 and 28% for females, respectively) and in males receiving 100 mg/kg/day (27% increase); high lymphocyte count in both sexes receiving 300 or 1000 mg/kg/day (increases of 21 and 32% for males and 34 and 31% increases for females, respectively); high large unstained cell counts in males receiving 100, 300 or 1000 mg/kg/day (all groups increased by 40%) and in females receiving 1000 mg/kg/day (50% increase); high monocyte counts in males receiving 100, 300 or 1000 mg/kg/day (20, 30 and 50% increases, respectively) and in females receiving 300 or 1000 mg/kg/day (13% increase in both groups) and high total leucocyte counts in both sexes receiving 300 or 1000 mg/kg/day (increases of 23 and 30% for males and 36 and 30% for females, respectively).

Treatment-related findings are summarised below:
Summary of treatment-related blood chemistry findings
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 1000 0 100 300 1000
Alkaline phosphatase (U/L) 92 112 127 171** 44 100 82 93
Bilirubin (µmol/L) 2 100* 150** 300** 2 100 100 100
Total cholesterol (mmol/L) 2.04 83** 71** 43** 1.74 103 97 94
High-density lipoprotein (mmol/L) 1.55 78** 66** 33** 1.51 102 96 91
Low-density lipoprotein (mmol/L) 0.22 109 109 68** 0.13 108 100 100
For controls, group means are shown. For treated groups, percent of controls are shown. Statistical significance is based on actual data (not on the percent differences).
* = p≤0.05; ** = p≤0.01
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination after 13 weeks revealed the following treatment-related findings: high alkaline phosphatase activity in males receiving 300 or 1000 mg/kg/day (increases of 27 and 71%, respectively); high bilirubin concentration in males receiving 300 or 1000 mg/kg/day (increases of 50 and 200%, respectively); low total cholesterol in males at all dose levels (83, 71 and 43% of control); low high density lipoprotein (HDL) concentrations in males at all dose levels (78, 66 and 33% of control) and reduced low-density lipoprotein concentration in males receiving 1000 mg/kg/day (68% of control).
Endocrine findings:
not specified
Description (incidence and severity):
The results from the analysis of serum samples for thyroid hormone levels are summarized below:
Summary of thyroid hormone concentrations (pg/mL) after 13 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 1000 0 100 300 1000
Triiodo-L-thyronine (T3) 697 799 800 776 782 979 953 850
Thyroxine (T4) 44700 49400 40700 30300** 34100 44300** 43900** 31400
Thyroid stimulating hormone (TSH) 844 968 1370 2610** 308 693* 958** 1710**
Significant when compared to Group 1: * - p<0.05; ** - p<0.01.
The analysis of serum thyroid hormone concentrations performed after 13 weeks revealed, when compared with controls, high serum thyroid stimulating hormone (TSH) concentrations in both sexes receiving 100, 300 or 1000 mg/kg/day (15, 62 and 209% increases in males and 125, 211 and 455% increases in females, respectively) with the extent of the difference from control being dose-related and attaining statistical significance in females at all dose levels and in males receiving 1000 mg/kg/day. In males, all individual values were within the background control range (117-8118 pg/mL; n=49) but in females 5/10 and 7/10 animals receiving 300 or 1000 mg/kg/day, respectively, had values about the background range (73-901 pg/mL; n=46).
There was, when compared with control, a slight decrease of thyroxine (T4) concentration in both sexes receiving 1000 mg/kg/day (68 and 92% of control in males and females, respectively) which was statistically significant only in males and all individual values were within the background range (33370-48518 pg/mL in males (n=50) and 27976-61536 pg/mL in females (n=49)). Triiodo L-thyronine (T3) concentrations were higher than control in all groups receiving tBuTPP Low TPP (15, 15 and 11% increases in males and 25, 22 and 9% increases in females receiving 100, 300 or 1000 mg/kg/day, respectively) but there was no dose-response, statistical significance was not attained and all indivudal values were within the background control
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ Weights

Toxicity phase animals
The analysis of organ weights performed after 13 weeks of treatment revealed a clearly treatment-related increase of absolute and body weight‑adjusted liver and kidney weights in both sexes receiving 100, 300 or 1000 mg/kg/day and increased body weight-adjusted thyroid gland weights in both sexes receiving 300 or 1000 mg/kg/day
Absolute and body weight-adjusted adrenal gland weights were higher than controls in males at all doses and in females receiving 1000 mg/kg/day and absolute and body weight-adjusted ovary gland weights were higher in females allocated to toxicity assessments receiving 1000 mg/kg/day.

Treatment related findings are summarized below:
Summary of treatment-related body-weight adjusted organ weight findings (g)
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 300 1000 0 100 300 1000
Liver 11.287 128** 146** 192** 8.326 116** 145** 221**
Thyroid gland 0.016 119 144** 163** 0.015 107 127* 187**
Kidney 1.929 115** 125** 129** 1.404 108* 112** 115**
Adrenal gland 0.050 122* 128** 144** 0.067 106 107 135**
Ovary NA NA NA NA 0.097 96 96 130*
For controls, group means are shown. For treated groups, percent of controls are shown. Statistical significance is based on actual data (not on the percent differences).
* = p≤0.05; ** = p≤0.01
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
no effects observed
Description (incidence and severity):
In the liver, abnormally dark coloration was noted in all animals administered 1000 mg/kg/day, 7 or 8/10 at 300 mg/kg/day and in a single male administered 100 mg/kg/day.

In the lungs, pale areas occurred in 3/10 animals of both sexes administered 1000 mg/kg/day and in 1/10 females administered 100 or 3/10 females administered 300 mg/kg/day.

Incidence of Test Article-Related Macroscopic Findings – Terminal Sacrifice After 13 Weeks of Treatment – Toxicity Phase Animals
tBuTPP Low TPP
Males Females
Dose Level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Liver
Number Examined 10 10 10 10 10 10 10 10
Abnormal Color, Dark 0 1 7 10 0 0 8 10
Lungs
Number Examined 10 10 10 10 10 10 10 10
Pale Area(s) 0 0 0 3 0 1 3 3

All other macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for this animal age, species and strain. Consequently, they were considered not test-item related.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related differences in the brain weights from perfusion-fixed neurotoxicity phase animals
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related non-adverse microscopic findings after 13 weeks of treatment occurred in the kidneys, liver, thyroid gland, lungs, adrenal glands and pancreas.

In the kidneys, minimal or slight tubular basophilia was present in controls and treated animals, with a higher incidence in males administered 1000 mg/kg/day. Tubular pigment was present in males administered 300 mg/kg/day and in both sexes administered 1000 mg/kg/day, at minimal severity. Slight cortical tubular hypertrophy, together with minimally increased eosinophilic inclusions, were present in males administered 1000 mg/kg/day, and minimal or slight cortical tubular hypertrophy and minimal increased eosinophilic inclusions in males administered 300 mg/kg/day.
One male administered 1000 mg/kg/day showed moderate tubular basophilia, tubular degeneration and granular casts which were likely due to tubular injury. These findings correlated macroscopically with bilateral depressions and abnormal kidney coloration present only in this animal. These findings were considered to be adverse.

In the liver, minimal to slight generalized hypertrophy was present in both sexes administered 100, 300 or 1000 mg/kg/day, with centrilobular hypertrophy present only in occasional animals administered 1000 mg/kg/day. This finding correlated with macroscopic finding of abnormal liver coloration and increased liver weights.

In the thyroid gland, minimal follicular cell hypertrophy was present in males administered 100 mg/kg/day and at minimal or slight severity in both sexes administered 300 or 1000 mg/kg/day, correlating with increased thyroid weights.

In the lungs, predominantly minimal alveolar macrophage aggregates were present in all groups, with a higher incidence in both sexes administered 300 or 1000 mg/kg/day. This finding was also seen in two control animals and correlated with macroscopic finding of pale areas of the lungs.

In the adrenal glands, minimal to slight cortical diffuse hypertrophy was present in both sexes administered 100, 300 or 1000 mg/kg/day, which correlated with increased adrenal gland weights.

In the pancreas, minimally to slightly decreased zymogen granules of acinar cells were reported in both sexes administered 1000 mg/kg/day.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cyclicity was unaffected by treatment. Examination of the testes revealed normal progression of the spermatogenic cycle.

Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Noe adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
Neurotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: tubular degeneration in the kidney
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
There was no evidence of a neurotoxic effect as determined by the neurobehavioral assessments, brain weight and neuropathology.
The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day in males and 1000 mg/kg/day in females. The NOAEL for neurotoxicity was 1000 mg/kg/day in both sexes.
Executive summary:

SUMMARY

The purpose of this study was to assess the neurotoxic effects and systemic toxic potential of tBuTPP Low TPP (an industrial chemical) when administered orally, by gavage, to Han Wistar rats for 13 weeks.

Three groups of 16 male and 16 female RccHan™;WIST rats received tBuTPP Low TPP at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. A similarly constituted control group received the vehicle, corn oil, at the same volume dose and over the same treatment period as treated groups.

During the study, clinical condition, neurobehavioral screening, body weight, food consumption, water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, estrous cycle, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken.

Results

There were no unscheduled deaths and the clinical appearance of the animals was unaffected by treatment. There were no treatment-related findings at the in-cage, in-hand or arena observations or at the reactivity and motor activity investigations.

Body weight gain was persistently lower than control from Week 2 in males receiving 1000 mg/kg/day (overall 16% reduction, Week 0-13) with no corresponding decrease of food intake. In females, all animals receivingtBuTPP Low TPP gained morebody weight than the controls during the first week of treatment, with the increased gain persisting throughout the study at 1000 mg/kg/day only (gain during Week 0-13 was 29% higher than control). This corresponded with a 21% overall increase of food intake in the animals. In males receiving 1000 mg/kg/day, water intake was 62 and 74% higher than control in males when quantified during Weeks 6 and 9, respectively and in females the increases were 74 and 77%, respectively. In males receiving 300 mg/kg/day, water intake was 21 and 29% higher than control in Weeks 6 and 9, respectively.

There were no treatment-related ophthalmoscopic findings.

The hematological examination after 13 weeks revealed the following treatment-related differences from control: high neutrophil count in both sexes receiving 300 or 1000 mg/kg/day (increases of 32 and 20% for males and 47 and 28% for females, respectively) and in males receiving 100 mg/kg/day (27% increase); high lymphocyte count in both sexes receiving 300 or 1000 mg/kg/day (increases of 21 and 32% for males and 34 and 31% increases for females, respectively); high large unstained cell counts in males receiving 100, 300 or 1000 mg/kg/day (all groups increased by 40%) and in females receiving 1000 mg/kg/day (50% increase); high monocyte counts in males receiving 100, 300 or 1000 mg/kg/day (20, 30 and 50% increases, respectively) and in females receiving 300 or 1000 mg/kg/day (13% increase in both groups) and high total leucocyte counts in both sexes receiving 300 or 1000 mg/kg/day (increases of 23 and 30% for males and 36 and 30% for females, respectively).

The biochemical examination after 13 weeks revealed the following treatment-related findings: high alkaline phosphatase activity in males receiving 300 or 1000 mg/kg/day (increases of 27 and 71%, respectively); high bilirubin concentration in males receiving 300 or 1000 mg/kg/day (increases of 50 and 200%, respectively); low total cholesterol in males at all dose levels (83, 71 and 43% of control); low high density lipoprotein (HDL) concentrations in males at all dose levels (78, 66 and 33% of control) and reduced low-density lipoprotein concentration in males receiving 1000 mg/kg/day (68% of control).

The analysis of serum thyroid hormone concentrations after 13 weeks revealed: high thyroid stimulating hormone (TSH) concentrations in both sexes receiving 100, 300 or 1000 mg/kg/day 15, 62 and 209% increases in males and 125, 211 and 455% increases in females, respectively); low thyroxine concentrations in both sexes receiving 1000 mg/kg/day (68 and 92% of control in males and females, respectively) and high triiodo L-thyronine concentrations in all groups receiving tBuTPP Low TPP (15, 15 and 11% increases in males and 25, 22 and 9% increases in females receiving 100, 300 or 1000 mg/kg/day, respectively) but there was no dose-response and statistical significance was not attained.

Estrous cyclicity was unaffected by treatment. Examination of the testes revealed normal progression of the spermatogenic cycle.

After 13 weeks of treatment liver and kidney weights were high in both sexes receiving 100, 300 or 1000 mg/kg/day and thyroid gland weights were high in both sexes receiving 300 or 1000 mg/kg/day. Adrenal gland weights were high in males at all doses and in females receiving 1000 mg/kg/day. Ovary gland weights were higher in females receiving 1000 mg/kg/day.

The macroscopic examination after 13 weeks revealed an increase in the incidence of abnormally dark colouration of the liver in both sexes receiving 300 or 1000 mg/kg/day and in males receiving 100 mg/kg/day and an increase in the incidence of pale areas on the lungs and bronchi in both sexes receiving 1000 mg/kg/day and in females receiving 300 mg/kg/day.

After 13 weeks, the administration of tBuTPP Low TPP was associated with non-adverse microscopic findings in the liver (hepatocyte hypertrophy),thyroid gland (follicular hypertrophy), lungs (alveolar macrophages aggregates), kidneys (tubular basophilia, tubular pigment, tubular cortical hypertrophy and increased eosinophilic inclusions), adrenal glands (diffuse cortical hypertrophy) and pancreas (decreased zymogen granules acinar cell) in animals receiving 100, 300 or 1000 mg/kg/day tBuTPP Low TPP allocated to the toxicity assessments. Renal tubular degeneration occurred in males receiving 1000 mg/kg/day and was adverse.

Conclusion

It is concluded that the oral administration oftBuTPP Low TPP to Han Wistar rats at doses up to 1000 mg/kg/day was associated with adverse findings in males at 1000 mg/kg/day which included reduced weight gain and histopathological findings oftubular degeneration in thekidney. There was evidence of a non-adverse, adaptive change in the liver (hepatocyte hypertrophyin the liver of both sexes at all dose levels and high plasma alkaline phosphatase activity and bilirubin concentration and lowplasma cholesterol concentration in males at all dose levels) which resulted in secondary rodent-specific hypertrophic changes in the follicular cells of the thyroid glands in both sexes at all dose levels, with the incidence and severity increasing with increasing dose, due to effects on serum thyroid hormones. Other non-adverse findings included increased leucocyte subpopulation counts in both sexes, mainly at 300 or 1000 mg/kg/day, adrenal gland cortical hypertrophy andalveolar macrophage aggregates in the lungs of both sexes at all dose levels and decreased zymogen granules of the pancreatic acinar cells in both sexes receiving 1000 mg/kg/day. Estrous cyclicity was unaffected by treatment and examination of the testes revealed normal progression of the spermatogenic cycle.

There was no evidence of a neurotoxic effect as determined by the neurobehavioral assessments, brain weight and neuropathology.

The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day in males and 1000 mg/kg/day in females. The NOAEL for neurotoxicity was 1000 mg/kg/day in both sexes.

 

Endpoint conclusion
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable Klimisch 1
Organ:
kidney

Additional information

In the key study for the oral route, Phosflex 51B was administered to rats in the diet. No treatment related mortality and clinical signs were noted in the animals. The statistically significant differences in hematology and clinical chemistry values and in red blood cell, plasma and brain cholinesterase activities between control and treated animals were minimal, inconsistent and considered not to be of biological significance. A biologically significant increase in liver and adrenal weights (only females) was noted in the high-dose groups, but this was not regarded as a toxic and therefore not an adverse effect. A NOAEL of 107.5 and 124.8 mg/kg bw/day (equivalent to 1600 ppm) was established for males and females, respectively.

Two supporting studies for the oral route were available: a 28-day and a 90-day toxicity study in rats. The subacute toxicity study (equivalent of established a NOAEL of 250 mg/kg bw/day based on liver effects in all dose groups, except for the lowest dose group. The 90-day toxicity study established a NOAEL of 71.6 mg/kg bw/day and 86.2 mg/kg bw/day for male and female, respectively, based on the absence of effects at the highest dose. The key study was chosen as such as it was performed using the most relevant exposure route (oral versus inhalation), a treatment period of 90 days (as compared to 28-days in the supporting study with a higher NOAEL) and it tested the highest concentration at which no adverse effects were observed (as compared to the other 90-day oral toxicity study).

Two subacute repeated dose toxicity studies for the dermal route are available. In the key study, rabbits were exposed to tBuTPP for 21 days. An evident dose response depression of terminal cholinesterase was observed in the treated males and females, which was significant in the mid and high dose. A NOAEL systemic of 10 mg/kg bw/day was derived. This finding was supported by a second 21 -day study in rabbits, in which a LOAEL systemic of 100 mg/kg bw/day was observed based on the same effects. Due to observed edema and desquamation at the low dose level in the key study, a NOAEL local could not be identified.

Additionally a 90-day inhalation toxicity study in rats was available that established a NOAEL systemic of 101.1 mg/m3 based on the absence of systemic effects at this dose (highest tested). A NOAEL local of 10.1 mg/m3 was established based on local effects on the respiratory system at the highest dose. These effects included rhinitis, sneezing and wheezing.

Justification for classification or non-classification

The most critical NOAEL established for t-BuTPP was 107.5 mg/kg bw/day. When considering the guidance values for classification as outlined in Annex I of 1272/2008/EC (significant effect observed at 100 mg/kg bw/day) and Annex VI of 67/548/EEC (significant effect observed at 50 mg/kg bw/day), the substance does not need to be classified for repeated dose toxicity.