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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined 28-day repeated dose toxicity with the reproduction/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2015 - April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2015
Deviations:
yes
Remarks:
procedures described in the section litter size
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 421 Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2015
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrabutylphosphonium bromide
EC Number:
221-487-8
EC Name:
Tetrabutylphosphonium bromide
Cas Number:
3115-68-2
Molecular formula:
C16H36P.Br
IUPAC Name:
tetrabutylphosphonium bromide
Test material form:
other: crystallized solid
Details on test material:
- Name of test material (as cited in study report): Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt
- Physical state: Light orange crystallized solid
- Storage condition of test material: At room temperature, container flushed with nitrogen
- Other: hygroscopic
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: WR150812A
- Expiration date of the batch: 08 August 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen; substance is hygroscopic

STABILITY IN VEHICLE
- propylene glycol; Stability for at least 6 hours at room temperature; 8 days in the refrigerator and 3 weeks in the freezer is confirmed over the concentration range 1 to 200 mg/mL

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: approximately 10-12 weeks; Females: approximately 12-14 weeks
- Weight at study initiation: 296-341 g for males and 209-242 f for females
- Fasting period before study: no
- Housing: Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages; Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages; Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages; Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages; Lactation: Pups were kept with the dam until termination in Macrolon plastic cages. General: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males)

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 November 2015 To: 08 April 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared within 8 days prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and in consultation with the Sponsor.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were exposed for 39-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 39-43 days.
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a dose range finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes, overnight (with a maximum of 24 hours)
- How many animals: 5 animals/sex/group
- Parameters according to guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13)
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

OTHER: Thyroid Hormone Analysis
F1-generation, PND 4 pups (from 2 surplus pups per litter at culling, if possible)
F1-generation, PND 13-15 pups (from 2 pups per litter (if possible from one male and one female) at planned necropsy
F0-generation, males and females, from all animals at planned necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to guidelines

HISTOPATHOLOGY: Yes, according to guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period that were considered related to treatment with the test item.
The weekly observations outside the home cage in a standard arena did not show any additional clinical signs of behavioural changes in any of the animals of all dose groups.
Salivation seen among animals of all groups receiving the test item during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence shortly after dosing. The severity of salivation showed a slight dose response relationship and also the presence of salivation after each dosing among the low dose group animals was less regular then among the animals of the mid and high dose group. An occasional presence of salivation among vehicle control animals was also noted.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (low dose) was found dead shortly after treatment on day 17 (day 3 of the mating period). Mainly based on the macroscopic findings, a dosing error may have been the cause of its spontaneous death. A normal body weight gain was observed in this female over the preceding two weeks, which did not suggest an affected health status of the animal in the period previous to its death. Its death was considered accidental and not to be related to treatment with the test item.
No further mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Slightly higher body weight gain was observed in the 30 mg/kg bw/day treated females during the lactation period, achieving a level of statistical significance on Day 4 of lactation when compared to vehicle controls. In the absence of a dose response relationship, this was considered an incidental finding and not related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
A lower food consumption in group 4 females was observed over days 0 to 4 of the post-coitum phase, achieving a level of statistical significance for the food consumption relative to body weight in comparison with controls. This low food consumption could mainly be attributed to a single female which showed a very low food consumption (and concurrent body weight gain) over this period. In the absence of any clinical signs in this animal and a further normal development of its pregnancy, this finding was considered of no toxicological significance. Slightly lower food consumption was observed in 300 mg/kg bw/day treated females during the lactation phase in comparison to the other groups. This difference may be related to the reduced litter size in these females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
A relatively high number of platelets was observed in the 100 mg/kg bw/day treated females, reaching a level of statistical significance when compared to vehicle controls. In the absence of a dose response relationship, this was considered an incidental finding and not related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- A dose related decrease for urea in both sexes, achieving levels of statistical significance in males of all three dose groups in comparison with controls.
- A dose related decrease for bile acids in males, achieving a level of statistical significance in 300 mg/kg bw/day treated males. In females, it should be noted that a high bile acid value was observed in one of the control females, significantly affecting the mean value (and the standard deviation) for this group. Exclusion of this value resulted in a group mean bile acid level of 23.9 (std.dev.12.7, n=4), indicating that the bile acids levels for the treated female groups were within the same range.
- A dose related increase for chloride in both sexes, achieving levels of statistical significance in males of all three dose groups and in 300 mg/kg bw/day treated females only.
- Increased creatinine in 300 mg/kg bw/day treated males, achieving a level of statistical significance.
No differences were noted in other clinical chemistry parameters between control and treated males and females.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
For grip strength, a dose related increase in grip strength of the fore limbs was observed in both males and females, achieving a level of statistical significance in the high dose group females, when compared to the vehicle controls. The magnitude of increase in the fore limb grip strength in the high dose animals was comparable in both sexes and was approximately 130-140 % in comparison with the concurrent control mean values. The cause of increase in grip strength could not be established from the results obtained in this study. However, the changes in grip strength were considered not to be an effect on the nervous system as neurological effects generally result in a decrease in grip strength. Despite the magnitude of increase in grip strength in high dose animals compared to that in controls in this study, the adversity of this finding up to the highest dose level was doubted, because all individual values observed in this study were within the range for grip strength values for untreated rats of this strain and age.
The hind limb grip strength mean values in males showed a large variation, achieving statistically significant differences in the low and mid dose males when compared to vehicle controls. These statistical significances were considered to have occurred as the result of relatively low grip strength values in controls and therefore of no toxicological relevance.
The hind limb grip strength mean values in females showed normal values.
For the motor activity, similar habituation profiles observed for all dose groups with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In 300 mg/kg bw/day treated males, a decreased mean weight of the prostate gland was found, reaching a level of statistical significance in comparison with control prostate gland weights.
In some of the other reproductive organs in males, i.e. the levator ani plus bulbocavernosus muscle complex (LABC), cowper’s gland, epididymides and seminal vesicles a (minimal) dose related decrease in weight was observed, both absolute and relative to body weight, achieving levels of statistical significance for the LABC in 300 mg/kg bw/day treated males only. It should be noted that all weight-values for the different organs, including those of the prostate gland, up to and including treatment at 300 mg/kg bw/day were within the normal range for male rats of this strain and age.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Tetrabutylphosphonium Bromide were noted in the stomach of the 300 mg/kg bw/day group males and females:
Stomach, increased apoptosis of the glandular mucosa was present in 1/5 males and 2/5 females at 300 mg/kg bw/day (minimal)
Stomach, edema of the glandular mucosa was present in 1/5 males at 300 mg/kg bw/day (minimal)
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Serum levels of T4 in F0 males (not determined in F0 females) showed a minimal tendency to decrease at higher dose levels. However, all mean T4 levels were well within the normal limits. Furthermore, no histopathological changes were found in the thyroid of the males. In the absence of any corroborative finding the changes in T4 levels were considered of no toxicological relevance.
Details on results:
Changes that were considered to be treatment-related were observed in grip strength, in some clinical chemistry parameters and organ weights and in the stomach.
A dose related increase in fore leg grip strength was observed in treated male and female rats, with a maximum increase of 130-140% in comparison with vehicle controls. The hind leg grip strength in these animals showed no deficits and no other corroborative findings were observed in the other (behavioural) parameters. The cause of the increase in grip strength could not be established from the results obtained in this study. However, the changes in grip strength were considered not to be an effect on the nervous system as neurological effects generally result in a decrease in grip strength. Despite the magnitude of increase in grip strength in high dose animals compared to that in controls in this study, the adversity of this finding up to the highest dose level was doubted, because all individual values observed in this study were within the range for grip strength values for untreated rats of this strain and age.
Chloride levels were significantly increased in both males and females at 300 mg/kg bw/day. The changes in this parameter in males at 30 and 100 mg/kg bw/day, also achieving a level of statistical significance at these dose levels in comparison with controls, were considered not adverse in nature as they were within the normal range.
In males treated at 300 mg/kg bw/day, increased levels of creatinine and decreased levels of bile acids were noted. In females, creatinine and bile acids were not affected by treatment.
A dose related decrease seemed apparent for urea in both males and females. As the decrease was only minimal and all values were within the normal range the changes were considered to be non-adverse in nature. Furthermore, no corroborative findings were found in any other study parameter that might explain the cause of the changes noted in these clinical chemistry parameters.
In males treated at 300 mg/kg bw/day, decreased prostate gland weights were observed and minimal dose related decreases in some of the other male reproductive organs, including the LABC, cowper’s gland, epididymides and seminal vesicles. Based on the magnitude of these changes and the fact that all weight-values for the different organs up to and including treatment at 300 mg/kg bw/day were within the normal range for male rats of this strain and age, these effects were considered to be non-adverse. Moreover, no histopathological changes were found in the prostate gland to support the prostate weight changes.
No parental toxicity was observed in the other organ weights in males and in any of the organ weights in females up to and including the highest dose level tested (300 mg/kg bw/day).
In males and females treated at 300 mg/kg bw/day, histopathological examination of the stomach revealed a minimal increased apoptosis of the glandular mucosa and/or edema of the glandular mucosa in a male and a few females.
No treatment-related changes were noted in the other parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, haematology investigations and macroscopic examinations).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
other: clinical biochemistry and non-neoplastic histopathology stomach
Organ:
other: clinical biochemistry and non-neoplastic histopathology stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

The accuracy of preparations was considered acceptable, with mean accuracy of 103-109-109% for the low-mid-high concentration, respectively, (criteria 90 -110% of the target concentration).

Homogeneity was 4.9% and 3.1% for the low and high concentration, respectively, demonstrating a coefficient of variation ≤ 10%.

Summary dose range finding study:

Group 1: 21 days, 150 mg/kg bw/day. During the second week of dosing hunched posture became apparent among the females, suggesting treatment related toxicity. Therefore, the treatment phase of this group was extended from 10 to 21 days. However, no (further) increase of signs of toxicity were observed up to 21 days of treatment in these animals.Liver and kidney weights considered to be normal.

Group 2: 10 days, 300 mg/kg bw/day. No clear signs of toxicity were observed among the females at this dose level. Liver and kidney weights considered to be normal.

Based on the results of this range finding study, dose levels for the main study were: 30, 100 and 300 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
A NOAEL for systemic toxicity of 100 mg/kg bw/day was derived for Tetrabutylphosphonium Bromide, based on observed changes in levels of clinical chemistry parameters and microscopic findings in the stomach at 300 mg/kg bw/day.
Executive summary:

Tetrabutylphosphonium Bromide was tested in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats by oral gavage according to OECD 422 Guideline (version July 2015) and in accordance with GLP principles. The dose levels for this study were 0, 30, 100 and 300 mg/kg bw/day. Males (10/dose) were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination, females (10/dose) were exposed for 39-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 39-43 days. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. One accidental death occurred in the study (a low dose female). No treatment-related mortality was observed. Chloride levels were significantly increased in both males and females at 300 mg/kg bw/day. The small increases in this parameter in males at 30 and 100 mg/kg bw/day were considered not adverse in nature as they were within the normal range. In males treated at 300 mg/kg bw/day, increased levels of creatinine and decreased levels of bile acids were noted. In females, creatinine and bile acids were not affected by treatment. A dose related decrease seemed apparent for urea in both males and females. As the decrease was only minimal and all values were within the normal range the changes were considered to be non -adverse in nature. In males and females treated at 300 mg/kg bw/day, histopathological examination of the stomach revealed a minimal increased apoptosis of the glandular mucosa and/or edema of the glandular mucosa in a few males and females. No treatment-related changes were noted in the other parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, haematology investigations and macroscopic examinations). Serum levels of T4 in F0 males (not determined in F0 females) showed a minimal tendency to decrease at higher dose levels. However, all mean T4 levels were well within the normal limits. Furthermore, no histopathological changes were found in the thyroid of the males. In the absence of any corroborative finding the change in T4 levels were considered of no toxicological relevance. In summary, treatment related changes that were considered to be toxicologically significant were observed in levels of chloride, creatinine and bile acids and in histopathological findings in the stomach. Based on these results, a NOAEL for systemic effects of 100 mg/kg bw/day was derived, based on changes in levels of clinical chemistry parameters and microscopic findings in the stomach at 300 mg/kg bw/day.

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