Tetrabutylphosphonium bromide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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

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

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

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.

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.

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).

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

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

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.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Devel opmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2015

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

October 2008

Deviations:

no

Test guideline 6

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

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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:

tetrabutylphosphanium 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 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 g for females
- Fasting period before study: no
- Housing: Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages; Premating: 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

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on 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%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

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.
Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week

Duration of test:

Until Day 13 after delivery

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

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

Maternal examinations:

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

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.

POST-MORTEM EXAMINATIONS: Yes
SACRIFICE
- Male animals: All surviving animals at the end of the exposure period of 29 Days
- Maternal animals: All surviving animals at least 13 Day after delivery (females which failed to deliver healthy offspring were sacrificed after 39-43 Days of exposure)
- Organs examined: according to Guidelines

OTHER:OTHER: Thyroid Hormone Analysis
F0-generation, males and females, from all animals at planned necropsy

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.

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.

Estrous cyclicity (parental animals)
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes, for females that failed to deliver healthy pups
- Number of late resorptions: Yes, for females that failed to deliver healthy pups

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

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.

Indices:

Reproductive indices
Mating index (%) = ((Number of females mated) / (Number of females paired)) * 100
Fertility index (%) = ((Number of pregnant females) / (Number of females paired)) * 100
Conception index (%) = ((Number of pregnant females) / (Number of females mated)) * 100
Gestation index (%) = ((Number of females bearing live pups) / (Number of pregnant females)) * 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices
Percentage live males at First Litter Check = ((Number of live male pups at First Litter Check) / (Number of live pups at First Litter Check)) * 100
Percentage live females at First Litter Check = ((Number of live female pups at First Litter Check) / (Number of live pups at First Litter Check)) * 100
Viability index (%) = ((Number of live offspring on Day 4 before culling) / (Number live offspring on Day 1 after littering)) * 100
Group mean values will be calculated from individual litter values.
Sex ratio (percentage males) = ((Number of males in litter) / Total number of offspring in litter) * 100
Lactation index (%) = ((Number of live offspring on Day 13 after littering) / (Number live offspring on Day 4 (after culling)) * 100
Group mean values were calculated from individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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 al bile acids levels for the treated 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), cow per’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, treatment-related

Description (incidence and severity):

T4 has not been determined in F0 females.

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 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).

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A lower number of implantation sites and corresponding lower number of pups per litter was observed at 300 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation was not considered to be affected by treatment
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation index and duration of gestation were not considered to be affected by treatment.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

The fertility and conception index were 100% (when excluding one female found dead on Day 3 of the mating period), 90% and 80% for the females treated at 30, 100 and 300 mg/kg bw/day, respectively.
One female at 100 mg/kg bw/day and two females at 300 mg/kg were not pregnant and one female at 100 mg/kgbw/day showed a resorption only.

Details on maternal toxic effects:

In 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 females. Chloride levels were significantly increased in females at 300 mg/kg bw day.
A lower number of implantation sites and corresponding lower number of pups per litter were observed in (pregnant) females treated at 300 mg/kg bw/day. Moreover, two females treated at 300 mg/kg bw/day appeared to be non-pregnant and one female which delivered only one (living) pup, had total litter loss on Day 2 of lactation. It could not be established from the results obtained in this study if the lower number of implantation sites observed in the females were indicative of effects on the fertility of the females or were related to effects on the fertility of the males.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

clinical biochemistry

histopathology: non-neoplastic

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

changes in levels of clinical chemistry parameters and microscopic findings in the stomach at 300 mg/kg bw/day,

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 1 (after littering) relative to the total number of offspring, or the live birth index, was 99, 97, 94 and 100% at 0, 30, 100 and 300 mg/kg bw/day, respectively, and did not show an effect of treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was not considered to be affected by treatment.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

A lower mean number of pups per litter was observed in the high dose group in comparison with vehicle controls.
The mean number of living pups per litter at first litter check on Day 1 of lactation was 11.0, 12.0, 10.3 and 8.3 at 0, 30, 100 and 300 mg/kg bw/day, respectively. It should be noted that one high dose female gave birth to only one pup.
The reduction in the mean number of living pups per litter in the high dose group, corresponds with the reduction in mean number of implantation sites seen in this group

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number off live offspring on Day 4 relative to that on Day 1 of lactation, or the viability index, was 99, 100, 100 and 95% at 0, 30, 100 and 300 mg/kg bw/day, respectively. The postnatal loss of living pups (pups missing) was 1, 0, 0 and 2 at 0, 30, 100 and 300 mg/kg bw/day. All pups went missing on Day 2 of lactation and no pups were found dead or missing between lactation Days 2 and 4. Pups missing were most likely cannibalised. The incidence and distribution of missing pups over the various dose groups did not indicate a relation to treatment and remained within the range considered normal for pups of this age.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
Autolysis was found in the majority of pups that were found dead.
Incidental macroscopic findings in pups at scheduled necropsy included swelling of a hind leg, scabs on the back and missing tail apex. These findings were all confirmations of observations made in the respective pups during the lactation period. Moreover, the small size of the pups in one litter, observed during the lactation period was also confirmed at necropsy for all pups.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
The statistical significance apparent in the normalized anogenital distance in female pups at 300 mg/kg bw/day was caused by the slightly higher body weights observed in these female pups on Day 1 of lactation. A slightly higher body weight of these pups might be related to the lower litter size of females in this high dose group. The slightly lower normalized anogenital distance in high dose pups was considered not directly related to treatment.

As for none of the examined male pups nipples were observed at PND 13, it was concluded that treatment with the test item up to and including 300 mg/kg bw/day had no effect on areola/nipple retention.

Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment.
The statistical significances apparent for T4 levels in the male PND 13-15 pups at 30 and 300 mg/kg bw/day were considered to be the result of relatively high control values in controls.

Details on embryotoxic / teratogenic effects:

A lower number of pups per litter were observed in the females treated at 300 mg/kg bw/day, which was considered to be the result of the lower number of implantation sites in these animals.
No treatment-related changes were noted in the remaining developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A lower number of pups per litter were observed in the females treated at 300 mg/kg bw/day, which was considered to be the result of the lower number of implantation sites in these animals.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

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:

The NOAEL for maternal toxicity for Tetrabutylphosphonium bromide is 100 mg/kg bw/day, based on observed changes of clinical chemistry parameters and microscopic findings in the stomach at 300 mg/kg bw/ day, and an observed lower number of implantation sites in females at 300 mg/kg bw/day.
A NOAEL for developmental toxicity of 100 mg/kg bw/day is derived based on reduced litter size, which is likely to be the result of the reproductive effects.

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.

A lower number of implantation sites and corresponding lower number of pups per litter were observed in (pregnant) females treated at 300 mg/kg bw/day. Moreover, two females treated at 300 mg/kg bw/day appeared to be non-pregnant and one female which delivered only one (living) pup, had total litter loss on Day 2 of lactation. It could not be established from the results obtained in this study if the lower number of implantation sites observed in the females were indicative of effects on the fertility of the females or were related to effects on the fertility of the males. At treatment at 100 mg/kg bw/day, one non-pregnant female was observed and one female with one resorption only. Pregnancy and delivery (of healthy pups) were not affected in control females and females treated at 30 mg/kg bw/day. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, precoital time, and estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Based on the observed lower number of implantation sites at 300 mg/kg bw/day, the NOAEL for reproductive toxicity is set at 100 mg/kg bw/day. A lower number of pups per litter were observed in the females treated at 300 mg/kg bw/day, which was considered to be the result of the lower number of implantation sites in these animals. No treatment-related changes were noted in the remaining developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/ nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy. Serum T4 levels in male and female PND 13-15 pups were not considered to be affected by treatment. The statistical significances apparent for T4 levels in the male PND 13-15 pups at 30 and 300 mg/kg bw/day were considered to be the result of relatively high control values in controls. Based on the observed reduced litter size at 300 mg/kg bw/day, which is likely the result of the reproductive effects, the NOAEL for developmental toxicity is 100 mg/kg bw/day.