Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2017 - 24 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2017 - 24 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 12-13 weeks old, females were 9-10 weeks old
- Weight at study initiation: males weighed between 267 and 304 g and females weighed between 209 and 238 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (Main females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were identically group-housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. In addition to pelleted rodent diet provided through a feed rack in the cover of the cage, several diet pellets were also
available in each cage with Group 4 males or females, as from Day 25 onwards. The diet pellets were replenished daily, if necessary.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. In addition to tap water in bottles in the cover of the cage, Hydrogel® 98% sterile water (ClearH2O, BioServices, Uden, The Netherlands) was also available in each cage with Group 4 males or females, as from Day 25 onwards. The hydrogel was replenished daily, if necessary.
- Acclimation period: 5 days prior to start of the pre-test period (Main females) or at least 5 days before the commencement of dosing (Main and Recovery males and Recovery females)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the object ives of the study.
Periodic analysis of the water is performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 46 to 72
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Oct 2017 To: 11 dec 2017
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose 1% (w/w), aqueous
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post coitum
- After successful mating each pregnant female was caged: individually in Macrolon plastic cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was = 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main and Recovery males (surviving up to the day of scheduled necropsy) were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were treated for 51-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. The female which failed to deliver or had a total litter loss was treated for 41 or 40 days respectively. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females.
Frequency of treatment:
daily
Dose / conc.:
7.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (with additional 5/sex/dose control and high dose recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of 2-(Diphenylphosphino) Benzoic Acid in rats, and in an attempt to produce graded responses to the test item.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first dosing and weekly thereafter. Mated main 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. Terminal body weight was recorded on the day of necropsy.

FOOD CONSUMPTION: yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: no

WATER CONSUMPTION: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: according to Guidelines

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 (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: According to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested. These tests were performed after dosing.
- Dose groups that were examined: all dose levels, selected 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static rightling reflex, fore- and hind limb grip strength, motor activity (total movements and ambulations)

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
weight of testis, epididymis, prostate gland, coagulation gland, seminal vesicles, and spermatogenic profiling
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males
and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma thyroid hormone level

GROSS EXAMINATION OF DEAD PUPS:
yes.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines
Postmortem examinations (offspring):
SACRIFICE
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. Suspected
treatment-related external abnormalities, i.e. eyes of pups showing white spot(s), were collected and fixed in 10% buffered formalin.
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in
10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 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) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection and/or hunched posture were regularly observed in several animals at 75 mg/kg bw/day from day 23 (in males) and day 19 (in females) onwards until the end of treatment. One recovery male showed continuous presence of hunched posture and piloerection during the recovery period from which it recovered 13 days after cessation of treatment. Additionally, lethargy was observed in 4/15 males at their final day of observation (two on day 28 before early sacrifice and two on day 29 (the day before scheduled sacrifice). Hypothermia and a lean appearance were additionally observed in two of the early sacrificed females on their final day of observation.
Salivation seen after dosing among animals of the 75 mg/kg bw/day dose group during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
No treatment related clinical signs were observed in male and female rats treated at 7.5 mg/kg bw/day and 25 mg/kg bw/day throughout the treatment period.
The incidental findings in a single control and high dose male comprising scabs on the head, ear or cheek occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three females and two males at 75 mg/kg bw/day (high dose) were sacrificed in extremis between Day 25 and 35 of treatment. On day 25 of treatment (Day 8 post-coitum), hypothermia and a lean appearance, supported by a 20% body weight loss compared to Day 0 post-coitum, were observed in one (main) female, which justified it sacrifice on that day. Since (first signs of) body weight loss were also seen in other high dose males and females, several diet pellets and hydrogel were provided at the bottom in each cage with high dose animals additionally to the feed and drinking water provided
in the cover of the cage from Day 25 onwards. These measures were taken to prevent starvation and dehydration as the result of a (possible) limited ability of the animals to reach out to the feed and water at the cover of the cage. In spite of this, a second main female had to be sacrificed on day 27 of treatment (Day 9 postcoitum) and a recovery female on day 35 of treatment, mainly because of considerably body weight loss of approximately 23% in both females. The two high dose males were sacrificed on day 28 of treatment. Lethargy was observed in both males on day 28 next to a body weight loss of 32% and 22%, respectively. Piloerection and hunched posture were the only clinical signs observed in all these high dose animals during a few days preceding the day of early sacrifice. One control female accidentally died immediately after blood sampling on the day of scheduled necropsy.
One female at 75 mg/kg bw/day was sacrificed within 24 hours after having given birth to a litter of dead pups (total litter loss) and another female at 75 mg/kg bw/day appeared to be non-pregnant and was sacrificed on post-coitum Day 26.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 75 mg/kg bw/day, the mean body weight gain was reduced from week 3 of treatment onwards, resulting in levels of statistical significance for body weights and body weight gain on Day 8 and Day 15 of the repro period when compared to control males. A high variation in changes in body weight was observed in the high dose males. Whereas some males still showed some (reduced) body weight gain, others lost considerable weight and had to be early sacrificed. This high variation was reflected in an increase standard deviation to the mean body weights and body weight gain for the high dose males. At the end of the 4-weeks treatment period the high dose males had on average gained only 14% of weight, whereas the average weight gain in the other groups was close to 30% compared to their weight at start of the study. After cessation of treatment, the recovery males all showed body weight gain but was still slightly lower in comparison with controls. At the end of the 14-day recovery period body weights of high dose male were still approximately 10% lower than that of control males.
Body weights and body weight gain of males treated at 7.5 and 25 mg/kg bw/day were similar to that of controls over the treatment period.
Body weights of treated pregnant (main) and recovery females remained in the same range as controls over the treatment period in all study phases. The presence of a single female with a high body weight in the recovery group resulted in a relatively high standard deviation to the mean body weights from day 8 of the repro period onwards. Since this female showed normal growth, no toxicological significance was attached to this finding.
One high dose female appeared to be non-pregnant and therefore its body weight and body weight gain was excluded from the summary tables. It body weights were considered to be normal and not affected by treatment.
Reduced body weight gain was observed on Day 4 and Day 7 post-coitum in females treated at 75 mg/kg bw/day in comparison with that in the other groups. The mean body weight values at these time points were affected by the body weight loss observed in two females, that were sacrificed in extremis between Day 7 and 11 post-coitum. Normal mean body weight gain was observed in the surviving (main) females from Day 11 post-coitum onwards.
After birth, 3/6 females at 75 mg/kg bw/day lost weight over days 0-4 of lactation, affecting the mean body weight gain for this dose group for Day 4 of lactation, and achieving a level of statistical significance in comparison with controls. On Day 13 of lactation, body weights of the females at 75 mg/kg bw/day had recovered, and the body weight gain was within the same range as controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Over the first two weeks of treatment, absolute and relative food consumption in males and females of treated groups was similar to the respective control levels. In 75 mg/kg bw/day (high dose) treated males, food consumption was lower during the repro period (weeks 3 and 4 of treatment) in comparison with that in the other groups. This was in line with the body weight loss seen in several males during this treatment period. The food consumption from week 4 onwards (days 8-15 of the repro–period) should be interpreted with care, because additional feed pellets were provided in all cages of the high dose animals which were not weighed and not counted for in determining the food consumption.
During the recovery period, the food consumption was still slightly lower in high dose males over the first week, but had recovered to similar values as in controls over the second week of the recovery period.
In females treated at 75 mg/kg bw/day, the food consumption was lower over days 0-4 and 4-7 postcoitum, achieving a level of statistical significance for absolute and relative food consumption when compared to controls over days 4-7, and slightly higher over days 11-14 post-coitum. This was in line with the body weight changes seen in several females during these time periods. The body weight loss in several lactating females over the first few days after delivery was also reflected by reduced food consumption over days 0-4 of lactation, achieving levels of statistical significance for absolute and relative food consumption when compared to controls. During the continuation of the lactation period, food consumption recovered but did not fully reach normal levels in high dose females before termination of the study.
The food consumption in (nulliparous) high dose recovery females was slightly lower from week 4 of treatment onwards in comparison with controls. During the recovery period the food consumption was similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- decreased reticulocyte counts in males and (nulliparous) recovery females (0.65x and 0.71x control, respectively), but not in lactating females,
- decreased red blood cell distribution width in males (RDW, 0.93x control),
- Increased haemoglobin and haematocrit concentrations in males (1.06x and 1.06x control). In addition, a comparable non-statistical significant increase in red blood cell count (1.05x control) was observed in males.
The haematological parameters of lactating (main) females up to 75 mg/kg bw/day, of main and recovery males and of recovery females up to and including 25 mg/kg bw/day were considered not to have been affected by treatment. In the absence of the dose-response relationship, the statistically significant differences in red blood cell count, haemoglobin and haematocrit arising between control and 25 mg/kg bw/day treated lactating (main) females were considered to have arisen by chance and not to represent a change of toxicological significance.
At the end of recovery, the reticulocytes count in high dose males and recovery females had recovered. The difference in RDW, haemoglobin and haematocrit values between high dose males and controls had diminished and the statistical significance had disappeared. The level of statistical significance present for MCV (p<0.05) in high dose recovery males was considered to have occurred by chance, because there is no indication of an effect in the haematocrit and/or red blood cell count from which the MCV is calculated. Since a similar difference was observed in red blood cell counts and haemoglobin levels between high dose recovery females and controls, and the calculated MCH values for both groups were comparable, the statistical significance for haemoglobin in recovery females at 75 mg/kg bw/day apparent at the end of the recovery period was considered a fortuitous finding and of no toxicological significance.
Coagulation parameters of treated male and female rats were considered not to have been affected by treatment. The PT and APTT values of treated rats were in the same range as the concurrent controls at the end of treatment as well as at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- Increased potassium levels in males (1.06x control),
- Decreased potassium levels in lactating females (0.9x control), but comparable potassium levels in control and 75 mg/kg bw/day treated (nulliparous) recovery females,
- Increased total protein concentration in lactating females (1.05x control),
- Decreased levels of total bilirubin, urea, creatine and chloride in (nulliparous) recovery females.
For interpretation of the changes in potassium levels in lactating females, it should be noted that the values obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/ kg bw/day were within the normal range (6).
Other statistically significant changes in clinical biochemistry parameters, i.e. decrease bile acids in 7.5 and 75 mg/kg bw/day males and inorganic phosphate in 7.5 mg/kg bw/day males, increased sodium in 25 mg/kg bw/day males and increased chloride in 7.5 and 25 mg/kg bw/day females, were considered to be not related to treatment as these occurred in the absence of a dose-related trend.
At the end of recovery, the difference in potassium levels between high dose males and controls had diminished and the statistical significance had disappeared. In recovery high dose females, recovery or diminishing of the differences was observed for the levels of total bilirubin, urea, creatine and chloride and the statistical significances had disappeared, except for creatine. Additionally, a statistical significance was apparent for the level of sodium in high dose recovery females when compared to controls at the end of recovery. However, the group-mean sodium values at the end of recovery were comparable and the difference between the two groups was even less than the difference at the end of treatment. Therefore, the statistical significance was the result of a very low variation in individual values within both groups and considered to have occurred by chance.
At the end of treatment, serum levels of T4 in treated F0 males were in the same range as in controls.

Historical control data (period 2015-2017):
Potassium (lactating females) - mean: 3.78, P5-P95:3.11-4.33 (n=186).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength in treated animals at all doses was comparable to that observed in the respective controls. Moreover, the grip strength between lactating and nulliparous females was similar in controls and females at 75 mg/kg bw/day. The variation in motor activity did not indicate a relation with treatment in main males and females. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The increase in activity towards the end of the measurement (interval 12) in the lactating (main) females is unexpected and cannot beexplained. Since it was observed in all four groups at a similar activity, no toxicological significance was attached to this finding.
In recovery males as well as in recovery (nulliparous) females a difference in motor activity between high dose (75 mg/kg bw/day) and control animals was noted, with higher values for total movements and ambulations in controls, not achieving statistically significant differences. In comparison with historical control data, the motor activity in control males was higher than expected, whereas that in the high dose males was within the normal range (5). In control and high dose, recovery females the total movements as well as the ambulations were within the normal range, although rather high in controls. Since the motor activity in high dose recovery males and females was within the normal range and no treatment related effect was observed in the main males and females, the differences in motor activity between controls and high dose recovery animals were considered to have occurred by change and of no toxicological significance.

(5) Historical control data for motor activity (period 2015-2017):
Total movements: males - mean: 3341 P5-P95:1734-5284 (n=200), females 4888 P5-P95:2507-8190 (n=369)
Ambulations: males - mean: 883 P5-P95:293-1143 (n=200), females 1337 P5-P95:627-2287 (n=359)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thyroid gland of males and females and are summarized in text table 2 and 3 (see `any other information on results incl tables`) and in the spleen, liver and bone marrow (femur and sternum) of males and are summarized in text table 4 (see `any other information on results incl tables`).
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in males starting at 25 mg/kg bw/day up to slight degree. After a 14-day treatment free period the follicular cell hypertrophy was still present at increased incidence and severity in males treated at 75 mg/kg bw/ day.
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in females starting at 7.5 mg/kg bw/day up to moderate degree. After a 14-day treatment free period the follicular cell hypertrophy showed complete recovery.
Spleen, a decreased incidence and severity of extramedullary hematopoiesis was present in males treated at 75 mg/kg bw/day. This finding correlated with the decreased organ weight. After a 14-day treatment free period the extramedullary hematopoiesis showed complete recovery.
Spleen, an increased incidence of pigmentation (likely hemosiderin) was present in males treated at 75 mg/kg bw/day. After a 14-day treatment free period the pigmentation (likely hemosiderin) showed complete recovery.
Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day up to slight degree. This finding correlated with the increased organ weight. After a 14-day treatment free period the hepatocellular hypertrophy showed complete recovery.
Bone marrow (sternum), atrophy was present in males treated at 75 mg/kg bw/day up to slight degree. After a 14-day treatment free period the atrophy showed complete recovery.
Bone marrow (femur), atrophy was present in males treated at 75 mg/kg bw/day at minimal degree.
After a 14-day treatment free period the atrophy showed complete recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not to be affected by treatment. All females showed evidence of mating.
Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 4 days after start of mating and the mean precoital time per group was 2.2, 1.7, 2.6 and 1.8 days for the control, 7.5, 25 and 75 mg/kg bw/day treated group, respectively.
Fertility index was considered not to be affected by treatment. All mated females were pregnant, except one female at 75 mg/kg bw/day. This single incidence of non-pregnancy after mating, which occasionally occurs in rats of this strain and age used in this type of studies, was considered a fortuitous, non-treatment-related finding also taking into account the absence of any treatment related finding in the other reproductive parameters in females of the high dose group.
Number of implantation sites was considered not to be affected by treatment. The mean number of implantation sites in pregnant females per group were 13.6, 13.2, 14.1 and 13.7 for the control, 7.5, 25 and 75 mg/kg bw/day treated group, respectively. The mean for the high dose group is including the implantations sites recorded for the two females that were sacrificed within 8-9 days after mating, but excluding the female that appeared to be non-pregnant.

Note: As two main female at 75 mg/kg bw/day were sacrificed early after mating during the post-coitum period, limited reproduction data are available for these two females in the high dose group.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
bone marrow
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
Absence of milk was seen in most pups that were found dead after the first litter check or preceding they were missing. This phenomenon is generally considered the cause of death as it is an indication that these pups were not able to feed themselves.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A litter of (fourteen) stillborn pups were recorded for one female at 75 mg/kg bw/day after 22 days of pregnancy. Retrospectively, the relatively low body weight gain of this female after mating until delivery was also considered indicative that the development of its offspring was affected. It should also be noted that the body weight of the female after delivery was more than 10% lower than at the moment of mating (207 g vs. 235 g).
In the six females at 75 mg/kg bw/day with living pups at birth, the live birth index was 100% and comparable to that in controls.
The live birth index in females treated at 7.5 and 25 mg/kg bw/day was considered not to be affected by treatment.
Four pups (in one litter) of the control group and a single pup 25 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

viability
From first litter check to culling (on PND 4), five pups (from two litters) of the 75 mg/kg bw/day group were found dead or missing. In the other groups, the incidence of dead/missing pups from first litter check to culling was 1, 2 (each from one litter) and 0 in the controls, 7.5 and 25 mg/kg bw/day groups, respectively. Pups missing were most likely cannibalised.

lactation index
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
One pup of the control group was sacrificed in extremis on PND 5 after showing laboured breathing and a dark red appearance with pale foci. A pup of the 75 mg/kg bw/day group was missing on PND 5, most likely cannibalised, without preceding signs. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range, for mortality between PND 4 to PND 13, considered normal for pups of this age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male and female pups per litter of the 75 mg/kg bw/day group were approximately 8% lower at birth in comparison with that of litters of controls. The difference in mean body weight remained comparable during lactation and was still approximately 8% on PND 13, achieving levels of statistical significance for male pups on PND 4 and for female pups on PND 4 and 7 during lactation.
The mean body weights of pups per litter of the 7.5 and 25 mg/kg bw/day groups were within the same range as that of control litters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-16 pups were considered not to be affected by treatment. In the absence of a doe-related response, the statistically significant difference in the mean T4 value apparent between the female pups at 7.5 mg/kg and controls was considered to have occurred by chance and of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In one or more pups in four (out of six) litters of the 75 mg/kg bw/day group, white spot(s) in one or both eyes were observed at macroscopic examination at sacrifice on PND 14-16.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after in utero and/or lactational exposure of F1 pups with 2-(Diphenylphosphino) Benzoic Acid of the 75 mg/kg bw/day group were noted in the eyes as shown in text table 5 (see 'any other information on results incl tables').
Eyes, degeneration lens fiber was present up to marked degree. This finding correlated with the macroscopic white spots;
Hyperplasia lens epithelium was present up to moderate degree;
Vacuolation lens fiber was present up to moderate degree;
Mineralization lens fiber was present up to slight degree;
Retinal folds were present up to moderate degree.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment. The sex ratios in litters per group were 47/53, 48/52, 48/52 and 40/60 for the control, 7.5, 25 and 75 mg/kg bw/day treated group, respectively.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 75 mg/kg bw/dayhad no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Key result
Dose descriptor:
LOAEL
Remarks:
based on lower body weight (during lactation) and alterations in the eyes of pups at 75 mg/kg bw/day; as eyes of pups from the 7.5 and 25 mg/kg bw/day groups were no investigated, a NOAEL for the observed alterations in the eyes could not be established
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
eye
Organ:
lens
retina
Treatment related:
yes
Relevant for humans:
yes
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
Treatment related:
no

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 91% and 96%). No test item was detected in the control group.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 1.2% and 1.8% in the low and high dose goups, respectively).

Note to Clinical signs, Body weight and Food consumption tables:

For Main group males, “Repro period” represents the mating phase. For Main females, “Repro period” represents the mating, post coitum and lactation phase. For Recovery group animals, the premating

period and Repro period represents the treatment phase.

Note to Functional tests, Haematology, Coagulation, Clinical chemistry and Organ weights tables:

Female Main and Recovery groups were evaluated separately because of the difference in physiological status, i.e. main females were lactating and recovery females were nulliparous.

Table 2.
Summary Test Item-Related Microscopic Findings – Thyroid Gland Males Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

5

5

5

5

5

5

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

1

3

2

3

2

2

      Slight           

-

-

2

1

-

2

a = Number of tissues examined from each group.

Table 3.Summary Test Item-Related Microscopic Findings – Thyroid Gland Females
Scheduled Euthanasia Animals

 

Main Females

Recovery Females

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

4

5

5

5

5

4

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

2

2

2

1

1

2

      Slight

-

2

2

2

-

-

      Moderate

-

-

   1

-

-

-

a = Number of tissues examined from each group.

Table 4.
Summary Test Item-Related Microscopic Findings – Spleen, Liver, Bone Marrow Males
Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

SPLEENa

5

5

5

5

5

5

   Extramedullary hematopoiesis

 

 

 

 

 

 

      Minimal

4

4

4

1

4

4

      Slight

1

1

1

-

1

-

 

 

 

 

 

 

 

   Pigmentation (likely hemosiderin)

 

 

 

 

 

 

      Minimal

1

1

1

4

2

2

 

 

 

 

 

 

 

LIVERa

4

5

5

5

5

5

   Hepatocellular hypertrophy

 

 

 

 

 

 

      Minimal

-

-

-

2

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (STERNUM)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (FEMUR)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

a = Number of tissues examined from each group.

Table 5. Summary Test Item-Related Microscopic Findings F1pups– Eyes

 

F1males

F1females

Dose level (mg/kg/day):

75

75

 

 

 

EYESa

5

6

   Degeneration lens fiber

 

 

      Minimal

-

1

      Slight

3

2

      Moderate

-

1

      Marked

2

2

   Hyperplasia lens epithelium

 

 

      Minimal

2

1

      Slight

-

1

      Moderate

2

2

   Vacuolation lens fiber

 

 

      Minimal

-

1

      Slight

3

2

      Moderate

2

3

   Mineralization lens fiber

 

 

      Minimal

1

-

      Slight

1

1

   Retinal fold

 

 

      Minimal

1

3

      Slight

1

1

      Moderate

1

-

a = Number of tissues examined from each group.

Conclusions:
Based on observed inncreased mortality, body weight changes, effect on red blood cell formation and histopathological changes in several organs, the NOAEL for systemic (parental) toxicity in this study is 25 mg/kg bw/day. The NOAEL for reproduction toxicity is at least 75 mg/kg bw/day (the highest dose tested).
For developmental toxicity a LOAEL of 75 mg/kg bw/day has been derived, based on lower body weight of the offspring during lactation and presence of alterations in the eyes among pups at 75 mg/kg bw/day; as eyes from high dose pups were investigated only, a definite NOAEL for the effects observed in the eyes could not be established).
Executive summary:

Wistar Han rats were treated with 2-(DIPHENYLPHOSPHINO) BENZOIC ACID by daily oral gavage at dose levels of 7.5, 25 and 75 mg/kg bw/day, followed by a 14-day treatment-free recovery period.

The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 14-16 days of lactation (for 51-56 days). Two females that failed to deliver (healthy) pups, i.e. a total litter loss and a non-pregnant female, were treated for 40 and 41 days, respectively.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

Salivation was noted among animals at 75 mg/kg bw/day during the treatment period. Taking into account the nature and minor severity of the salivation and its time of occurrence (i.e. after dosing), this sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

From week 3 of treatment onwards, sudden body weight loss was observed among males and females treated at 75 mg/kg bw/day. Between Days 25 and 35, three females and two males had to be sacrificed, mainly because of severe body weight loss. All these decedents showed piloerection and hunched posture and in some of them also hypothermia (one female) or lethargy (two males) was observed shortly before early sacrifice. In several animals surviving to scheduled necropsy, piloerection and/or hunched posture was also regularly observed from week 3 onwards. Additionally, lethargy was seen in two males on the day before scheduled sacrifice. In males at 75 mg/kg bw/day, the individual changes in body weight gain from week 3 of treatment onwards resulted in lower mean body weights in males at 75 mg/kg bw/day in comparison with controls. In 75 mg/kg bw/day treated main females, reduced body weight gain was observed over the first week post-coitum, and again over the first week of lactation, but no effects on body weight were observed in the non-mated, recovery females. Changes in food consumption in both sexes observed during the study were in line with the changes in body weight.

At the end of treatment, changes in haematology parameters were noted in males and (nulliparous) recovery females at 75 mg/kg bw/day only and consisted of decreased reticulocyte counts and red blood cell distribution width. In addition, increased red blood cell counts and haemoglobin and haematocrit concentrations were observed in males at the end of treatment. The combination of these changes might indicate an effect on the formation of red blood cells and, as a result, the proportion of young and old erythrocytes in the peripheral blood might have been altered. Despite the effects on red blood cells in the peripheral blood, no alterations were observed in the bone marrow after histopathological examination in these animals. In lactating females at 75 mg/kg bw/day, no treatment-related changes in haematology parameters were observed at the end of treatment. At the end of the 14-Day recovery period, the differences in haematology parameters between the high dose animals and controls had diminished or disappeared.

At the end of treatment, changes in clinical biochemistry parameters were noted in males, lactating and (nulliparous) recovery females at 75 mg/kg bw/day. Increased potassium levels were observed in males and decreased potassium levels in lactating females. The toxicological significance of the latter was doubted, since the potassium levels obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/kg bw/day were within the normal range. In addition, no effect on potassium was observed in high dose recovery females at the end of treatment. Other changes at the end of treatment were observed as increased total protein in lactating females and decreased total bilirubin, urea, creatine and chloride in recovery females. Since all changes in clinical biochemistry parameters in male and female rats treated at 75 mg/kg bw/day were within the historical control range for rats of this strain and age, they were considered minimal and non-adverse in nature. At the end of treatment, serum levels of T4 in treated F0-males were in the same range as in controls.

There were five test item-related deaths in the high dose group. No definite cause of death could be determined for these animals. For one decedent, a stomach ulceration was considered to be the cause of moribundity.

There were no test item-related macroscopic findings.

There were test item-related decreases in thymus and spleen weight (absolute) and an increase in liver (relative to body weight) in main group males treated at 75 mg/kg bw/day. There was no microscopic correlate for the organ weight changes in the thymus. Test item-related microscopic findings were present in:

-Thyroid gland, follicular cell hypertrophy was present at slightly increased incidence and severity in females starting at 7.5 mg/kg bw/day and in males starting at 25 mg/kg bw/day. After the 14-day treatment-free period the slight increase in incidence and severity was still present in males, but had completely recovered in females.

-Spleen, extramedullary hematopoiesis was present at decreased incidence and severity in males treated at 75 mg/kg bw/day. This correlated with the decrease in organ weight. After the 14-day treatment-free period there was complete recovery. Pigmentation (likely hemosiderin) was present at increased incidence in males treated at 75 mg/kg bw/day. After the 14-day treatment-free period there was complete recovery.

-Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day. This correlated with the increase in organ weight. After the 14-day treatment-free period there was complete recovery.

-Bone marrow (femur and sternum), atrophy was present in males treated at 75 mg/kg bw/day/day. After the 14-day treatment-free period there was complete recovery.

After the 14-day treatment-free period there was an increase in spleen weight (only relative to body weight) in recovery group males at 75 mg/kg bw/day. There was no microscopic correlate to this. No signs of toxicity were observed in the parental male and female rats treated at 7.5 and 25 mg/kg bw/day.

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (75 mg/kg bw/day). It should be noted that two main females at 75 mg/kg bw/day were sacrificed moribund 8-9 days postcoitum. These two females appeared to be pregnant and their number of implantations sites were included in the evaluation of the reproductive results. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

As a result of the sacrifice of two (pregnant) females at 75 mg/kg bw/day in the post-coitum period, no developmental data were available for these two females in the high dose group. In addition, one mated female appeared to be not pregnant and another delivered a complete litter of stillborn pups (regarded as total litter loss). The (single) incidence of these latter findings was within normal background range for rats of this strain and age and in this type of studies, but due to the fact that they both were present in the high dose group, the results on development could only be evaluated for six litters, compared to for ten litters in the other groups.

In the six high dose litters obtained in this study, a relatively high number of 5 unaccounted for sites (the difference in number of implantation sites and number of pups born) was observed in one female and a low viability index (the pup loss between PND1 to PND4) was obtained for the litter of another female. Again, the single incidence of these findings does occasionally occurs among rats of this strain and age and in this type of studies. A possible relation between the single incidences of total litter loss, a high number of unaccounted for sites and a high post-natal pup-loss could not be established in this study, but they could all be considered an effect on development of the pups. Taking into account that a high variability in treatment-related effects was observed among high dose females, some high dose females seemed unaffected by treatment whereas others were sacrificed moribund, the variation in developmental effects in the offspring might be the result of maternal toxicity.

Other developmental effects were observed as a lower body weights of the male and female pups in litters at 75 mg/kg bw/day during lactation. The body weights were approximately 8% lower from birth to PND 13. Furthermore, white spot(s) were observed in one or more pups in four (out of six) litters at macroscopic examination. Microscopic examination of these eyes from the high dose pups demonstrated degeneration of lens fiber (up to marked degree). This finding correlated with the macroscopic white spots. Furthermore hyperplasia lens epithelium was present (up to moderate degree), vacuolation of lens fiber (up to moderate degree), mineralization lens fiber (up to mild degree) and retinal folds were present (up to moderate degree). As only eyes with white spots from the high dose group were included in the histopathological investigation, no information is available on the eyes of the mid and low dose pups.

No treatment-related changes were noted in the other developmental parameters investigated in this study (i.e. gestation, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, lactation index, clinical signs, anogenital distance, areola/nipple retention and T4 thyroid hormone levels.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL)/ lowest-observed-adverse-effect level (LOAEL) of 2-(DIPHENYLPHOSPHINO) BENZOIC ACID were established:

Parental NOAEL: 25 mg/kg bw/day (based on treatment related mortality, body weight changes, effects on red blood cell formation and histopathological changes in several organs at 75 mg/kg bw/day)

Reproduction NOAEL: at least 75 mg/kg bw/day

Developmental LOAEL: 75 mg/kg bw/day (based on lower body weight of the offspring during lactation and presence of alterations in the eyes among pups at 75 mg/kg bw/day; as eyes from high dose pups were investigated only, a definite NOAEL for the effects observed in the eyes could not be established)

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2017 - 24 January 2018 Reliability 1
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
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: 606
- Expiration date of the lot/batch: 22 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature desiccated
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 12-13 weeks old, females were 9-10 weeks old
- Weight at study initiation: males weighed between 267 and 304 g and females weighed between 209 and 238 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (Main females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were identically group-housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. In addition to pelleted rodent diet provided through a feed rack in the cover of the cage, several diet pellets were also available in each cage with Group 4 males or females, as from Day 25 onwards. The diet pellets we re replenished daily, if necessary.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. In addition to tap water in bottles in the cover of the cage, Hydrogel® 98% sterile water (ClearH2O, BioServices, Uden, The Netherlands) was also available in each cage with Group 4 males or females, as from Day 25 onwards. The hydrogel was replenished daily, if necessary.
- Acclimation period: 5 days prior to start of the pre-test period (Main females) or at least 5 days before the commencement of dosing (Main and Recovery males and Recovery females)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the object ives of the study.
Periodic analysis of the water is performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 46 to 72
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Oct 2017 To: 11 dec 2017
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose 1% (w/w), aqueous
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was = 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main and Recovery males (surviving up to the day of scheduled necropsy) were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were treated for 51-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. The female which failed to deliver or had a total litter loss was treated for 41 or 40 days respectively. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females.
Frequency of treatment:
daily
Dose / conc.:
7.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (with additional 5/sex/dose control and high dose recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of 2-(Diphenylphosphino) Benzoic Acid in rats, and in an attempt to produce graded responses to the test item.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first dosing and weekly thereafter. Mated main 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. Terminal body weight was recorded on the day of necropsy.

FOOD CONSUMPTION: yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: no

WATER CONSUMPTION: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: according to Guidelines

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 (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: According to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested. These tests were performed after dosing.
- Dose groups that were examined: all dose levels, selected 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static rightling reflex, fore- and hind limb grip strength, motor activity (total movements and ambulations)

IMMUNOLOGY: No

Sacrifice and pathology
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations
F0 generation: Estrous cycle determination, cohabitating/mating procedure, general reproduction data, TSH and total T4 measurement.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males
and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma thyroid hormone level

GROSS EXAMINATION OF DEAD PUPS:
yes.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 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) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection and/or hunched posture were regularly observed in several animals at 75 mg/kg bw/day from day 23 (in males) and day 19 (in females) onwards until the end of treatment. One recovery male showed continuous presence of hunched posture and piloerection during the recovery period from which it recovered 13 days after cessation of treatment. Additionally, lethargy was observed in 4/15 males at their final day of observation (two on day 28 before early sacrifice and two on day 29 (the day before scheduled sacrifice). Hypothermia and a lean appearance were additionally observed in two of the early sacrificed females on their final day of observation.
Salivation seen after dosing among animals of the 75 mg/kg bw/day dose group during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
No treatment related clinical signs were observed in male and female rats treated at 7.5 mg/kg bw/day and 25 mg/kg bw/day throughout the treatment period.
The incidental findings in a single control and high dose male comprising scabs on the head, ear or cheek occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three females and two males at 75 mg/kg bw/day (high dose) were sacrificed in extremis between Day 25 and 35 of treatment. On day 25 of treatment (Day 8 post-coitum), hypothermia and a lean appearance, supported by a 20% body weight loss compared to Day 0 post-coitum, were observed in one (main) female, which justified it sacrifice on that day. Since (first signs of) body weight loss were also seen in other high dose males and females, several diet pellets and hydrogel were provided at the bottom in each cage with high dose animals additionally to the feed and drinking water provided in the cover of the cage from Day 25 onwards. These measures were taken to prevent starvation and dehydration as the result of a (possible) limited ability of the animals to reach out to the feed and water at the cover of the cage. In spite of this, a second main female had to be sacrificed on day 27 of treatment (Day 9 postcoitum) and a recovery female on day 35 of treatment, mainly because of considerably body weight loss of approximately 23% in both females. The two high dose males were sacrificed on day 28 of treatment. Lethargy was observed in both males on day 28 next to a body weight loss of 32% and 22%, respectively. Piloerection and hunched posture were the only clinical signs observed in all these high dose animals during a few days preceding the day of early sacrifice. One control female accidentally died immediately after blood sampling on the day of scheduled necropsy.
One female at 75 mg/kg bw/day was sacrificed within 24 hours after having given birth to a litter of dead pups (total litter loss) and another female at 75 mg/kg bw/day appeared to be non-pregnant and was sacrificed on post-coitum Day 26.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 75 mg/kg bw/day, the mean body weight gain was reduced from week 3 of treatment onwards, resulting in levels of statistical significance for body weights and body weight gain on Day 8 and Day 15 of the repro period when compared to control males. A high variation in changes in body weight was observed in the high dose males. Whereas some males still showed some (reduced) body weight gain, others lost considerable weight and had to be early sacrificed. This high variation was reflected in an increase standard deviation to the mean body weights and body weight gain for the high dose males. At the end of the 4-weeks treatment period the high dose males had on average gained only 14% of weight, whereas the average weight gain in the other groups was close to 30% compared to their weight at start of the study. After cessation of treatment, the recovery males all showed body weight gain but was still slightly lower in comparison with controls. At the end of the 14-day recovery period body weights of high dose male were still approximately 10% lower than that of control males.
Body weights and body weight gain of males treated at 7.5 and 25 mg/kg bw/day were similar to that of controls over the treatment period.
Body weights of treated pregnant (main) and recovery females remained in the same range as controls over the treatment period in all study phases. The presence of a single female with a high body weight in the recovery group resulted in a relatively high standard deviation to the mean body weights from day 8 of the repro period onwards. Since this female showed normal growth, no toxicological significance was attached to this finding.
One high dose female appeared to be non-pregnant and therefore its body weight and body weight gain was excluded from the summary tables. It body weights were considered to be normal and not affected by treatment.
Reduced body weight gain was observed on Day 4 and Day 7 post-coitum in females treated at 75 mg/kg bw/day in comparison with that in the other groups. The mean body weight values at these time points were affected by the body weight loss observed in two females, that were sacrificed in extremis between Day 7 and 11 post-coitum. Normal mean body weight gain was observed in the surviving (main) females from Day 11 post-coitum onwards.
After birth, 3/6 females at 75 mg/kg bw/day lost weight over days 0-4 of lactation, affecting the mean body weight gain for this dose group for Day 4 of lactation, and achieving a level of statistical significance in comparison with controls. On Day 13 of lactation, body weights of the females at 75 mg/kg bw/day had recovered, and the body weight gain was within the same range as controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Over the first two weeks of treatment, absolute and relative food consumption in males and females of treated groups was similar to the respective control levels. In 75 mg/kg bw/day (high dose) treated males, food consumption was lower during the repro period (weeks 3 and 4 of treatment) in comparison with that in the other groups. This was in line with the body weight loss seen in several males during this treatment period. The food consumption from week 4 onwards (days 8-15 of the repro–period) should be interpreted with care, because additional feed pellets were provided in all cages of the high dose animals which were not weighed and not counted for in determining the food consumption.
During the recovery period, the food consumption was still slightly lower in high dose males over the first week, but had recovered to similar values as in controls over the second week of the recovery period.
In females treated at 75 mg/kg bw/day, the food consumption was lower over days 0-4 and 4-7 postcoitum, achieving a level of statistical significance for absolute and relative food consumption when compared to controls over days 4-7, and slightly higher over days 11-14 post-coitum. This was in line with the body weight changes seen in several females during these time periods. The body weight loss in several lactating females over the first few days after delivery was also reflected by reduced food consumption over days 0-4 of lactation, achieving levels of statistical significance for absolute and relative food consumption when compared to controls. During the continuation of the lactation period, food consumption recovered but did not fully reach normal levels in high dose females before termination of the study.
The food consumption in (nulliparous) high dose recovery females was slightly lower from week 4 of treatment onwards in comparison with controls. During the recovery period the food consumption was similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- decreased reticulocyte counts in males and (nulliparous) recovery females (0.65x and 0.71x control, respectively), but not in lactating females,
- decreased red blood cell distribution width in males (RDW, 0.93x control),
- Increased haemoglobin and haematocrit concentrations in males (1.06x and 1.06x control). In addition, a comparable non-statistical significant increase in red blood cell count (1.05x control) was observed in males.
The haematological parameters of lactating (main) females up to 75 mg/kg bw/day, of main and recovery males and of recovery females up to and including 25 mg/kg bw/day were considered not to have been affected by treatment. In the absence of the dose-response relationship, the statistically significant differences in red blood cell count, haemoglobin and haematocrit arising between control and 25 mg/kg bw/day treated lactating (main) females were considered to have arisen by chance and not to represent a change of toxicological significance.
At the end of recovery, the reticulocytes count in high dose males and recovery females had recovered. The difference in RDW, haemoglobin and haematocrit values between high dose males and controls had diminished and the statistical significance had disappeared. The level of statistical significance present for MCV (p<0.05) in high dose recovery males was considered to have occurred by chance, because there is no indication of an effect in the haematocrit and/or red blood cell count from which the MCV is calculated. Since a similar difference was observed in red blood cell counts and haemoglobin levels between high dose recovery females and controls, and the calculated MCH values for both groups were comparable, the statistical significance for haemoglobin in recovery females at 75 mg/kg bw/day apparent at the end of the recovery period was considered a fortuitous finding and of no toxicological significance.
Coagulation parameters of treated male and female rats were considered not to have been affected by treatment. The PT and APTT values of treated rats were in the same range as the concurrent controls at the end of treatment as well as at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- Increased potassium levels in males (1.06x control),
- Decreased potassium levels in lactating females (0.9x control), but comparable potassium levels in control and 75 mg/kg bw/day treated (nulliparous) recovery females,
- Increased total protein concentration in lactating females (1.05x control),
- Decreased levels of total bilirubin, urea, creatine and chloride in (nulliparous) recovery females.
For interpretation of the changes in potassium levels in lactating females, it should be noted that the values obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/ kg bw/day were within the normal range (6).
Other statistically significant changes in clinical biochemistry parameters, i.e. decrease bile acids in 7.5 and 75 mg/kg bw/day males and inorganic phosphate in 7.5 mg/kg bw/day males, increased sodium in 25 mg/kg bw/day males and increased chloride in 7.5 and 25 mg/kg bw/day females, were considered to be not related to treatment as these occurred in the absence of a dose-related trend.
At the end of recovery, the difference in potassium levels between high dose males and controls had diminished and the statistical significance had disappeared. In recovery high dose females, recovery or diminishing of the differences was observed for the levels of total bilirubin, urea, creatine and chloride and the statistical significances had disappeared, except for creatine. Additionally, a statistical significance was apparent for the level of sodium in high dose recovery females when compared to controls at the end of recovery. However, the group-mean sodium values at the end of recovery were comparable and the difference between the two groups was even less than the difference at the end of treatment. Therefore, the statistical significance was the result of a very low variation in individual values within both groups and considered to have occurred by chance.
At the end of treatment, serum levels of T4 in treated F0 males were in the same range as in controls.

Historical control data (period 2015-2017):
Potassium (lactating females) - mean: 3.78, P5-P95:3.11-4.33 (n=186).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength in treated animals at all doses was comparable to that observed in the respective controls. Moreover, the grip strength between lactating and nulliparous females was similar in controls and females at 75 mg/kg bw/day. The variation in motor activity did not indicate a relation with treatment in main males and females. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The increase in activity towards the end of the measurement (interval 12) in the lactating (main) females is unexpected and cannot be explained. Since it was observed in all four groups at a similar activity, no toxicological significance was attached to this finding.
In recovery males as well as in recovery (nulliparous) females a difference in motor activity between high dose (75 mg/kg bw/day) and control animals was noted, with higher values for total movements and ambulations in controls, not achieving statistically significant differences. In comparison with historical control data, the motor activity in control males was higher than expected, whereas that in the high dose males was within the normal range (5). In control and high dose, recovery females the total movements as well as the ambulations were within the normal range, although rather high in controls. Since the motor activity in high dose recovery males and females was within the normal range and no treatment related effect was observed in the main males and females, the differences in motor activity between controls and high dose recovery animals were considered to have occurred by change and of no toxicological significance.

(5) Historical control data for motor activity (period 2015-2017):
Total movements: males - mean: 3341 P5-P95:1734-5284 (n=200), females 4888 P5-P95:2507-8190 (n=369)
Ambulations: males - mean: 883 P5-P95:293-1143 (n=200), females 1337 P5-P95:627-2287 (n=359)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a test item-related decrease in thymus and spleen weight (only absolute, -24% and -15%, respectively) and an increase in liver weight (only relative to body weight, +12%) in main group male s treated at 75 mg/kg bw/day. In the recovery group, there was an increase in spleen weight (only relative to body weight, +13%) in males treated at 75 mg/kg bw/day.
Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a decrease in final body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were five test item-related deaths in animals, two males and three females, treated at 75 mg/kg bw/day. Four of these decedents were emaciated. Additionally, a reduced size of the thymus (in one male) and dark red foci in the glandular stomach at necropsy (in one female) were observed in single animals at necropsy. One male showed no macroscopic findings.
There were no test item-related gross observations in all other animals (surviving to scheduled necropsy) of all groups. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thyroid gland of males and females and are summarized in text table 2 and 3 (see `any other information on results incl tables`) and in the spleen, liver and bone marrow (femur and sternum) of males and are summarized in text table 4 (see `any other information on results incl tables`).
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in males starting at 25 mg/kg bw/day up to slight degree. After a 14-day treatment free period the follicular cell hypertrophy was still present at increased incidence and severity in males treated at 75 mg/kg bw/ day.
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in females starting at 7.5 mg/kg bw/day up to moderate degree. After a 14-day treatment free period the follicular cell hypertrophy showed complete recovery.
Spleen, a decreased incidence and severity of extramedullary hematopoiesis was present in males treated at 75 mg/kg bw/day. This finding correlated with the decreased organ weight. After a 14-day treatment free period the extramedullary hematopoiesis showed complete recovery.
Spleen, an increased incidence of pigmentation (likely hemosiderin) was present in males treated at 75 mg/kg bw/day. After a 14-day treatment free period the pigmentation (likely hemosiderin) showed complete recovery.
Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day up to slight degree. This finding correlated with the increased organ weight. After a 14-day treatment free period the hepatocellular hypertrophy showed complete recovery.
Bone marrow (sternum), atrophy was present in males treated at 75 mg/kg bw/day up to slight degree. After a 14-day treatment free period the atrophy showed complete recovery.
Bone marrow (femur), atrophy was present in males treated at 75 mg/kg bw/day at minimal degree.
After a 14-day treatment free period the atrophy showed complete recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
no effects on reproduction was observed
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly low post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) of 86% was observed at 75 mg/kg bw/day, compared with 91%, 94 and 91% in the controls and 7.5 and 25 mg/kg bw/day groups, respectively. The individual difference in number of implantation sites and number of pups born (the so-called unaccounted for (implantation) sites) in each treated group was within the same range as in controls, i.e. 0-4, except for one female at 75 mg/kg bw/day with 5 unaccounted for sites. Because of the low number of eight high dose females, compared to ten in the other groups, the contribution of the 5 unaccounted for sites, a not uncommon finding in female rats of this strain and age in this type of studies, to the calculated post-survival implantation index of this group is relatively high. Therefore, no toxicological significance was attached to the changes in the post-implantation survival index observed in the current study.
For one female the number of pups was higher than the number of implantations, i.e 16 pups compared to 15 implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
A litter of (fourteen) stillborn pups were recorded for one female at 75 mg/kg bw/day after 22 days of pregnancy. Retrospectively, the relatively low body weight gain of this female after mating until delivery was also considered indicative that the development of its offspring was affected. It should also be noted that the body weight of female no.88 after delivery was more than 10% lower than at the moment of mating (207 g vs. 235 g).
In the six females at 75 mg/kg bw/day with living pups at birth, the live birth index was 100% and comparable to that in controls.
The live birth index in females treated at 7.5 and 25 mg/kg bw/day was considered not to be affected by treatment.
Four pups (in one litter) of the control group and a single pup 25 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. The gestation index was 100% (n=10) for the control and 7.5 and 25 mg/kg bw/day treated females and 88% (n=8) for the 75 mg/.kg bw/day treated females. The two high dose females that were sacrificed before littering were not included in the calculation of the gestation index. The mean duration of gestation per group was 21.5, 21.3, 21.5 and 21.6 for the control, 7.5, 25 and 75 mg/kg bw/day treated group, respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment. All mated females were pregnant, except one female at 75 mg/kg bw/day. This single incidence of non-pregnancy after mating, which occasionally occurs in rats of this strain and age used in this type of studies, was considered a fortuitous, non-treatment-related finding also taking into account the absence of any treatment related finding in the other reproductive parameters in females of the high dose group.
Other effects:
no effects observed
Description (incidence and severity):
no effects on reproduction was observed
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
haematology
histopathology: non-neoplastic
Key result
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
treatment related mortality, body weight changes, effects on red blood cell formation and histopathological changes in several organs
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male and female pups per litter of the 75 mg/kg bw/day group were approximately 8% lower at birth in comparison with that of litters of controls. The difference in mean body weight remained comparable during lactation and was still approximately 8% on PND 13, achieving levels of statistical significance for male pups on PND 4 and for female pups on PND 4 and 7 during lactation.
The mean body weights of pups per litter of the 7.5 and 25 mg/kg bw/day groups were within the same range as that of control litters.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
A litter of (fourteen) stillborn pups were recorded for one female at 75 mg/kg bw/day after 22 days of pregnancy. Retrospectively, the relatively low body weight gain of this female after mating until delivery was also considered indicative that the development of its offspring was affected. It should also be noted that the body weight of the female after delivery was more than 10% lower than at the moment of mating (207 g vs. 235 g).
In the six females at 75 mg/kg bw/day with living pups at birth, the live birth index was 100% and comparable to that in controls.
The live birth index in females treated at 7.5 and 25 mg/kg bw/day was considered not to be affected by treatment.
Four pups (in one litter) of the control group and a single pup 25 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.


Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment. The sex ratios in litters per group were 47/53, 48/52, 48/52 and 40/60 for the control, 7.5, 25 and 75 mg/kg bw/day treated group, respectively.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
A lower mean number of living pups at first litter check was recorded at 75 mg/kg bw/day (9.7 vs.12.0 in the control group). It should be noted that this mean value was largely affected by one high dose female with a complete litter of stillborn pups. Excluding this female, a mean value of 11.3 was calculated for the number of living pups in females at 75 mg/kg bw/day (n=6), and comparable with the mean value in controls.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
From first litter check to culling (on PND 4), five pups (from two litters) of the 75 mg/kg bw/day group were found dead or missing. In the other groups, the incidence of dead/missing pups from first litter check to culling was 1, 2 (each from one litter) and 0 in the controls, 7.5 and 25 mg/kg bw/day groups, respectively. Pups missing were most likely cannibalised.

lactation index
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
One pup of the control group was sacrificed in extremis on PND 5 after showing laboured breathing and a dark red appearance with pale foci. A pup of the 75 mg/kg bw/day group was missing on PND 5, most likely cannibalised, without preceding signs. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range, for mortality between PND 4 to PND 13, considered normal for pups of this age.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
In one or more pups in four (out of six) litters of the 75 mg/kg bw/day group, white spot(s) in one or both eyes were observed at macroscopic examination at sacrifice on PND 14-16. The eyes from F1 pups (from dams treated at 75 mg/kg/day only) with macroscopic white spots noted at necropsy, were examined histologically.
The nature and incidence of the other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were present in the eye(s) of male and female F1 pups of the 75 mg/kg/day treated group:
-Degeneration lens fiber was present up to marked degree. This finding correlated with the macroscopic white spots;
-Hyperplasia lens epithelium was present up to moderate degree;
-Vacuolation lens fiber up to moderate degree;
-Mineralization lens fiber was present up to mild degree;
Retinal folds were present up to moderate degree.
Only eyes of pups observed with white spots during macroscopic examination were investigated microscopically.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 75 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 13-16 pups were considered not to be affected by treatment. In the absence of a doe-related response, the statistically significant difference in the mean T4 value apparent between the female pups at 7.5 mg/kg bw/day and controls was considered to have occurred by chance and of no toxicological significance.
Key result
Dose descriptor:
LOAEL
Remarks:
based on lower body weight (during lactation) and alterations in the eyes of pups at 75 mg/kg bw/day; as eyes of pups from the 7.5 and 25 mg/kg bw/day groups were no investigated, a NOAEL for the observed alterations in the eyes could not be established.
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: alterations in the eyes of pups at 75 mg/kg bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
visceral/soft tissue: eye
Description (incidence and severity):
observations were in 5 males and 6 females:
degeneration of lens fiber (up to marked) degree, hyperplasia of lens epithelium (upt o moderate degree), vacuolation of lens fiber (up to moderate degree), mineralization of lens fiber (up to mild degree), retinal folds (up to moderate degree)
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Relevant for humans:
yes

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 91% and 96%). No test item was detected in the control group.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 1.2% and 1.8% in the low and high dose goups, respectively).

Note to Clinical signs, Body weight and Food consumption tables:

For Main group males, “Repro period” represents the mating phase. For Main females, “Repro period” represents the mating, post coitum and lactation phase. For Recovery group animals, the premating

period and Repro period represents the treatment phase.

Note to Functional tests, Haematology, Coagulation, Clinical chemistry and Organ weights tables:

Female Main and Recovery groups were evaluated separately because of the difference in physiological status, i.e. main females were lactating and recovery females were nulliparous.

Table 2.
Summary Test Item-Related Microscopic Findings – Thyroid Gland Males Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

5

5

5

5

5

5

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

1

3

2

3

2

2

      Slight           

-

-

2

1

-

2

a = Number of tissues examined from each group.

Table 3.Summary Test Item-Related Microscopic Findings – Thyroid Gland Females
Scheduled Euthanasia Animals

 

Main Females

Recovery Females

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

4

5

5

5

5

4

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

2

2

2

1

1

2

      Slight

-

2

2

2

-

-

      Moderate

-

-

   1

-

-

-

a = Number of tissues examined from each group.

Table 4.
Summary Test Item-Related Microscopic Findings – Spleen, Liver, Bone Marrow Males
Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

SPLEENa

5

5

5

5

5

5

   Extramedullary hematopoiesis

 

 

 

 

 

 

      Minimal

4

4

4

1

4

4

      Slight

1

1

1

-

1

-

 

 

 

 

 

 

 

   Pigmentation (likely hemosiderin)

 

 

 

 

 

 

      Minimal

1

1

1

4

2

2

 

 

 

 

 

 

 

LIVERa

4

5

5

5

5

5

   Hepatocellular hypertrophy

 

 

 

 

 

 

      Minimal

-

-

-

2

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (STERNUM)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (FEMUR)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

a = Number of tissues examined from each group.

Table 5. Summary Test Item-Related Microscopic Findings F1pups– Eyes

 

F1males

F1females

Dose level (mg/kg/day):

75

75

 

 

 

EYESa

5

6

   Degeneration lens fiber

 

 

      Minimal

-

1

      Slight

3

2

      Moderate

-

1

      Marked

2

2

   Hyperplasia lens epithelium

 

 

      Minimal

2

1

      Slight

-

1

      Moderate

2

2

   Vacuolation lens fiber

 

 

      Minimal

-

1

      Slight

3

2

      Moderate

2

3

   Mineralization lens fiber

 

 

      Minimal

1

-

      Slight

1

1

   Retinal fold

 

 

      Minimal

1

3

      Slight

1

1

      Moderate

1

-

a = Number of tissues examined from each group.

Conclusions:
Based on observed inncreased mortality, body weight changes, effect on red blood cell formation and histopathological changes in several organs, the NOAEL for systemic (parental) toxicity in this study is 25 mg/kg bw/day. The NOAEL for reproduction toxicity is at least 75 mg/kg bw/day (the highest dose tested).
For developmental toxicity a LOAEL of 75 mg/kg bw/day has been derived, based on lower body weight of the offspring during lactation and presence of alterations in the eyes among pups at 75 mg/kg bw/day; as eyes from high dose pups were investigated only, a definite NOAEL for the effects observed in the eyes could not be established).
Executive summary:

Wistar Han rats were treated with 2-(DIPHENYLPHOSPHINO) BENZOIC ACID by daily oral gavage at dose levels of 7.5, 25 and 75 mg/kg bw/day, followed by a 14-day treatment-free recovery period.

The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 14-16 days of lactation (for 51-56 days). Two females that failed to deliver (healthy) pups, i.e. a total litter loss and a non-pregnant female, were treated for 40 and 41 days, respectively.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

Salivation was noted among animals at 75 mg/kg bw/day during the treatment period. Taking into account the nature and minor severity of the salivation and its time of occurrence (i.e. after dosing), this sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

From week 3 of treatment onwards, sudden body weight loss was observed among males and females treated at 75 mg/kg bw/day. Between Days 25 and 35, three females and two males had to be sacrificed, mainly because of severe body weight loss. All these decedents showed piloerection and hunched posture and in some of them also hypothermia (one female) or lethargy (two males) was observed shortly before early sacrifice. In several animals surviving to scheduled necropsy, piloerection and/or hunched posture was also regularly observed from week 3 onwards. Additionally, lethargy was seen in two males on the day before scheduled sacrifice. In males at 75 mg/kg bw/day, the individual changes in body weight gain from week 3 of treatment onwards resulted in lower mean body weights in males at 75 mg/kg bw/day in comparison with controls. In 75 mg/kg bw/day treated main females, reduced body weight gain was observed over the first week post-coitum, and again over the first week of lactation, but no effects on body weight were observed in the non-mated, recovery females. Changes in food consumption in both sexes observed during the study were in line with the changes in body weight.

At the end of treatment, changes in haematology parameters were noted in males and (nulliparous) recovery females at 75 mg/kg bw/day only and consisted of decreased reticulocyte counts and red blood cell distribution width. In addition, increased red blood cell counts and haemoglobin and haematocrit concentrations were observed in males at the end of treatment. The combination of these changes might indicate an effect on the formation of red blood cells and, as a result, the proportion of young and old erythrocytes in the peripheral blood might have been altered. Despite the effects on red blood cells in the peripheral blood, no alterations were observed in the bone marrow after histopathological examination in these animals. In lactating females at 75 mg/kg bw/day, no treatment-related changes in haematology parameters were observed at the end of treatment. At the end of the 14-Day recovery period, the differences in haematology parameters between the high dose animals and controls had diminished or disappeared.

At the end of treatment, changes in clinical biochemistry parameters were noted in males, lactating and (nulliparous) recovery females at 75 mg/kg bw/day. Increased potassium levels were observed in males and decreased potassium levels in lactating females. The toxicological significance of the latter was doubted, since the potassium levels obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/kg bw/day were within the normal range. In addition, no effect on potassium was observed in high dose recovery females at the end of treatment. Other changes at the end of treatment were observed as increased total protein in lactating females and decreased total bilirubin, urea, creatine and chloride in recovery females. Since all changes in clinical biochemistry parameters in male and female rats treated at 75 mg/kg bw/day were within the historical control range for rats of this strain and age, they were considered minimal and non-adverse in nature. At the end of treatment, serum levels of T4 in treated F0-males were in the same range as in controls.

There were five test item-related deaths in the high dose group. No definite cause of death could be determined for these animals. For one decedent, a stomach ulceration was considered to be the cause of moribundity.

There were no test item-related macroscopic findings.

There were test item-related decreases in thymus and spleen weight (absolute) and an increase in liver (relative to body weight) in main group males treated at 75 mg/kg bw/day. There was no microscopic correlate for the organ weight changes in the thymus. Test item-related microscopic findings were present in:

-Thyroid gland, follicular cell hypertrophy was present at slightly increased incidence and severity in females starting at 7.5 mg/kg bw/day and in males starting at 25 mg/kg bw/day. After the 14-day treatment-free period the slight increase in incidence and severity was still present in males, but had completely recovered in females.

-Spleen, extramedullary hematopoiesis was present at decreased incidence and severity in males treated at 75 mg/kg bw/day. This correlated with the decrease in organ weight. After the 14-day treatment-free period there was complete recovery. Pigmentation (likely hemosiderin) was present at increased incidence in males treated at 75 mg/kg bw/day. After the 14-day treatment-free period there was complete recovery.

-Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day. This correlated with the increase in organ weight. After the 14-day treatment-free period there was complete recovery.

-Bone marrow (femur and sternum), atrophy was present in males treated at 75 mg/kg bw/day/day. After the 14-day treatment-free period there was complete recovery.

After the 14-day treatment-free period there was an increase in spleen weight (only relative to body weight) in recovery group males at 75 mg/kg bw/day. There was no microscopic correlate to this. No signs of toxicity were observed in the parental male and female rats treated at 7.5 and 25 mg/kg bw/day.

Reproductive results

No reproduction toxicity was observed up to and including the highest dose level tested (75 mg/kg bw/day). It should be noted that two main females at 75 mg/kg bw/day were sacrificed moribund 8-9 days postcoitum. These two females appeared to be pregnant and their number of implantations sites were included in the evaluation of the reproductive results. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

As a result of the sacrifice of two (pregnant) females at 75 mg/kg bw/day in the post-coitum period, no developmental data were available for these two females in the high dose group. In addition, one mated female appeared to be not pregnant and another delivered a complete litter of stillborn pups (regarded as total litter loss). The (single) incidence of these latter findings was within normal background range for rats of this strain and age and in this type of studies, but due to the fact that they both were present in the high dose group, the results on development could only be evaluated for six litters, compared to for ten litters in the other groups.

In the six high dose litters obtained in this study, a relatively high number of 5 unaccounted for sites (the difference in number of implantation sites and number of pups born) was observed in one female and a low viability index (the pup loss between PND1 to PND4) was obtained for the litter of another female. Again, the single incidence of these findings does occasionally occurs among rats of this strain and age and in this type of studies. A possible relation between the single incidences of total litter loss, a high number of unaccounted for sites and a high post-natal pup-loss could not be established in this study, but they could all be considered an effect on development of the pups. Taking into account that a high variability in treatment-related effects was observed among high dose females, some high dose females seemed unaffected by treatment whereas others were sacrificed moribund, the variation in developmental effects in the offspring might be the result of maternal toxicity.

Other developmental effects were observed as a lower body weights of the male and female pups in litters at 75 mg/kg bw/day during lactation. The body weights were approximately 8% lower from birth to PND 13. Furthermore, white spot(s) were observed in one or more pups in four (out of six) litters at macroscopic examination. Microscopic examination of these eyes from the high dose pups demonstrated degeneration of lens fiber(up to marked degree). This finding correlated with the macroscopic white spots. Furthermore hyperplasia lens epitheliumwas present (up to moderate degree), vacuolation of lens fiber (up to moderate degree), mineralization lens fiber(up to mild degree) and retinal foldswere present (up to moderate degree). As only eyes with white spots from the high dose group were included in the histopathological investigation, no information is available on the eyes of the mid and low dose pups.

No treatment-related changes were noted in the other developmental parameters investigated in this study (i.e. gestation, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, lactation index, clinical signs, anogenital distance, areola/nipple retention and T4 thyroid hormone levels.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL)/ lowest-observed-adverse-effect level (LOAEL) of 2-(DIPHENYLPHOSPHINO) BENZOIC ACID were established:

Parental NOAEL: 25 mg/kg bw/day (based on treatment related mortality, body weight changes, effects on red blood cell formation and histopathological changes in several organs at 75 mg/kg bw/day)

Reproduction NOAEL: at least 75 mg/kg bw/day

Developmental LOAEL: 75 mg/kg bw/day (based on lower body weight of the offspring during lactation and presence of alterations in the eyes among pups at 75 mg/kg bw/day; as eyes from high dose pups were investigated only, a definite NOAEL for the effects observed in the eyes could not be established)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: pale yellow powder, free flowing crystalline
- Storage conditions: at room temperature desiccated

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 12-13 weeks old, females were 9-10 weeks old
- Weight at study initiation: males weighed between 267 and 304 g and females weighed between 209 and 238 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (Main females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were identically group-housed during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. In addition to pelleted rodent diet provided through a feed rack in the cover of the cage, several diet pellets were also available in each cage with Group 4 males or females, as from Day 25 onwards. The diet pellets were replenished daily, if necessary.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. In addition to tap water in bottles in the cover of the cage, Hydrogel® 98% sterile water (ClearH2O, BioServices, Uden, The Netherlands) was also available in each cage with Group 4 males or females, as from Day 25 onwards. The hydrogel was replenished daily, if necessary.
- Acclimation period: 5 days prior to start of the pre-test period (Main females) or at least 5 days before the commencement of dosing (Main and Recovery males and Recovery females)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 46 to 72
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Oct 2017 To: 11 dec 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
methylcellulose
Remarks:
1% (w/w), aqueous
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was = 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main and Recovery males (surviving up to the day of scheduled necropsy) were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were treated for 51-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. The female which failed to deliver or had a total litter loss was treated for 41 or 40 days respectively. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
7.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (with additional 5/sex/dose control and high dose recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of 2-(Diphenylphosphino) Benzoic Acid in rats, and in an attempt to produce graded responses to the test item.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first dosing and weekly thereafter. Mated main 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. Terminal body weight was recorded on the day of necropsy.

FOOD CONSUMPTION: yes
Food consumption was quantitatively measured weekly, except for males and females which
were housed together for mating and for females without evidence of mating. Food
consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum
and during lactation on PND 1, 4, 7, and 13.


FOOD EFFICIENCY: no

WATER CONSUMPTION: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: according to Guidelines

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 (Main and recovery F0-males and recovery F0-females; Main F0-females were not fasted) overnight for maximum 24 hours; water was available.
- How many animals: selected 5 animals/sex/group
- Parameters examined: According to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13), and all Recovery females were tested on the first day a Main female was tested. These tests were performed after dosing.
- Dose groups that were examined: all dose levels, selected 5 animals/sex/group
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static rightling reflex, fore- and hind limb grip strength, motor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER: for details on reproductive and developmental toxicity examinations see sections 7.8.1 and 7.8.2 (cross-references)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines
Other examinations:
F0 generation: Estrous cycle determination, cohabitating/mating procedure, general reproduction data, TSH and total T4 measurement
F1 generation: mortality, clinical observations, BW, sex, AGD, areola/nipple retention, total T4 measurement
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection and/or hunched posture were regularly observed in several animals at 75 mg/kg bw/day from day 23 (in males) and day 19 (in females) onwards until the end of treatment. One recovery male showed continuous presence of hunched posture and piloerection during the recovery period from which it recovered 13 days after cessation of treatment. Additionally, lethargy was observed in 4/15 males at their final day of observation (two on day 28 before early sacrifice and two on day 29 (the day before scheduled sacrifice). Hypothermia and a lean appearance were additionally observed in two of the early sacrificed females on their final day of observation.
Salivation seen after dosing among animals of the 75 mg/kg bw/day dose group during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
No treatment related clinical signs were observed in male and female rats treated at 7.5 mg/kg bw/day and 25 mg/kg bw/day throughout the treatment period.
The incidental findings in a single control and high dose male comprising scabs on the head, ear or cheek occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three females and two males at 75 mg/kg bw/day (high dose) were sacrificed in extremis between Day 25 and 35 of treatment. On day 25 of treatment (Day 8 post-coitum), hypothermia and a lean appearance, supported by a 20% body weight loss compared to Day 0 post-coitum, were observed in one (main) female, which justified it sacrifice on that day. Since (first signs of) body weight loss were also seen in other high dose males and females, several diet pellets and hydrogel were provided at the bottom in each cage with high dose animals additionally to the feed and drinking water provided in the cover of the cage from Day 25 onwards. These measures were taken to prevent starvation and dehydration as the result of a (possible) limited ability of the animals to reach out to the feed and water at the cover of the cage. In spite of this, a second main female had to be sacrificed on day 27 of treatment (Day 9 postcoitum) and a recovery female on day 35 of treatment, mainly because of considerably body weight loss of approximately 23% in both females. The two high dose males were sacrificed on day 28 of treatment. Lethargy was observed in both males on day 28 next to a body weight loss of 32% and 22%, respectively. Piloerection and hunched posture were the only clinical signs observed in all these high dose animals during a few days preceding the day of early sacrifice. One control female accidentally died immediately after blood sampling on the day of scheduled necropsy.
One female at 75 mg/kg bw/day was sacrificed within 24 hours after having given birth to a litter of dead pups (total litter loss) and another female at 75 mg/kg bw/day appeared to be non-pregnant and was sacrificed on post-coitum Day 26.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 75 mg/kg bw/day, the mean body weight gain was reduced from week 3 of treatment onwards, resulting in levels of statistical significance for body weights and body weight gain on Day 8 and Day 15 of the repro period when compared to control males. A high variation in changes in body weight was observed in the high dose males. Whereas some males still showed some (reduced) body weight gain, others lost considerable weight and had to be early sacrificed. This high variation was reflected in an increase standard deviation to the mean body weights and body weight gain for the high dose males. At the end of the 4-weeks treatment period the high dose males had on average gained only 14% of weight, whereas the average weight gain in the other groups was close to 30% compared to their weight at start of the study. After cessation of treatment, the recovery males all showed body
weight gain but was still slightly lower in comparison with controls. At the end of the 14-day recovery period body weights of high dose male were still approximately 10% lower than that of control males.
Body weights and body weight gain of males treated at 7.5 and 25 mg/kg bw/day were similar to that of controls over the treatment period.
Body weights of treated pregnant (main) and recovery females remained in the same range as controls over the treatment period in all study phases. The presence of a single female with a high body weight in the recovery group resulted in a relatively high standard deviation to the mean body weights from day 8 of the repro period onwards. Since this female showed normal growth, no toxicological significance was attached to this finding.
One high dose female appeared to be non-pregnant and therefore its body weight and body weight gain was excluded from the summary tables. It body weights were considered to be normal and not affected by treatment.
Reduced body weight gain was observed on Day 4 and Day 7 post-coitum in females treated at 75 mg/kg bw/day in comparison with that in the other groups. The mean body weight values at these time points were affected by the body weight loss observed in two females, that were sacrificed in extremis between Day 7 and 11 post-coitum. Normal mean body weight gain was observed in the surviving (main) females from Day 11 post-coitum onwards.
After birth, 3/6 females at 75 mg/kg bw/day lost weight over days 0-4 of lactation, affecting the mean body weight gain for this dose group for Day 4 of lactation, and achieving a level of statistical significance in comparison with controls. On Day 13 of lactation, body weights of the females at 75 mg/kg bw/day had recovered, and the body weight gain was within the same range as controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Over the first two weeks of treatment, absolute and relative food consumption in males and females of treated groups was similar to the respective control levels. In 75 mg/kg bw/day (high dose) treated males, food consumption was lower during the repro period (weeks 3 and 4 of treatment) in comparison with that in the other groups. This was in line with the body weight loss seen in several males during this treatment period. The food consumption from week 4 onwards (days 8-15 of the repro–period) should be interpreted with care, because additional feed pellets were provided in all cages of the high dose animals which were not weighed and not counted for in determining the food consumption.
During the recovery period, the food consumption was still slightly lower in high dose males over the first week, but had recovered to similar values as in controls over the second week of the recovery period.
In females treated at 75 mg/kg bw/day, the food consumption was lower over days 0-4 and 4-7 postcoitum, achieving a level of statistical significance for absolute and relative food consumption when compared to controls over days 4-7, and slightly higher over days 11-14 post-coitum. This was in line with the body weight changes seen in several females during these time periods. The body weight loss in several lactating females over the first few days after delivery was also reflected by reduced food consumption over days 0-4 of lactation, achieving levels of statistical significance for absolute and relative food consumption when compared to controls. During the continuation of the lactation period, food consumption recovered but did not fully reach normal levels in high dose females before termination of the study.
The food consumption in (nulliparous) high dose recovery females was slightly lower from week 4 of treatment onwards in comparison with controls. During the recovery period the food consumption was similar to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- decreased reticulocyte counts in males and (nulliparous) recovery females (0.65x and 0.71x control, respectively), but not in lactating females,
- decreased red blood cell distribution width in males (RDW, 0.93x control),
- Increased haemoglobin and haematocrit concentrations in males (1.06x and 1.06x control). In addition, a comparable non-statistical significant increase in red blood cell count (1.05x control) was observed in males.
The haematological parameters of lactating (main) females up to 75 mg/kg bw/day, of main and recovery males and of recovery females up to and including 25 mg/kg bw/day were considered not to have been affected by treatment. In the absence of the dose-response relationship, the statistically significant differences in red blood cell count, haemoglobin and haematocrit arising between control and 25 mg/kg bw/day treated lactating (main) females were considered to have arisen by chance and not to represent a change of toxicological significance.
At the end of recovery, the reticulocytes count in high dose males and recovery females had recovered. The difference in RDW, haemoglobin and haematocrit values between high dose males and controls had diminished and the statistical significance had disappeared. The level of statistical significance present for MCV (p<0.05) in high dose recovery males was considered to have occurred by chance, because there is no indication of an effect in the haematocrit and/or red blood cell count from which the MCV is calculated. Since a similar difference was observed in red blood cell counts and haemoglobin levels between high dose recovery females and controls, and the calculated MCH values for both groups were comparable, the statistical significance for haemoglobin in recovery females at 75 mg/kg bw/day apparent at the end of the recovery period was considered a fortuitous finding and of no toxicological significance.
Coagulation parameters of treated male and female rats were considered not to have been affected by treatment. The PT and APTT values of treated rats were in the same range as the concurrent controls at the end of treatment as well as at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistical significant changes distinguished males and/or females at 75 mg/kg bw/day from control animals at the end of treatment:
- Increased potassium levels in males (1.06x control),
- Decreased potassium levels in lactating females (0.9x control), but comparable potassium levels in control and 75 mg/kg bw/day treated (nulliparous) recovery females,
- Increased total protein concentration in lactating females (1.05x control),
- Decreased levels of total bilirubin, urea, creatine and chloride in (nulliparous) recovery females.
For interpretation of the changes in potassium levels in lactating females, it should be noted that the values obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/kg bw/day were within the normal range (6).
Other statistically significant changes in clinical biochemistry parameters, i.e. decrease bile acids in 7.5 and 75 mg/kg bw/day males and inorganic phosphate in 7.5 mg/kg bw/day males, increased sodium in 25 mg/kg bw/day males and increased chloride in 7.5 and 25 mg/kg bw/day females, were considered to be not related to treatment as these occurred in the absence of a dose-related trend.
At the end of recovery, the difference in potassium levels between high dose males and controls had diminished and the statistical significance had disappeared. In recovery high dose females, recovery or diminishing of the differences was observed for the levels of total bilirubin, urea, creatine and chloride and the statistical significances had disappeared, except for creatine. Additionally, a statistical significance was apparent for the level of sodium in high dose recovery females when compared to controls at the end of recovery. However, the group-mean sodium values at the end of recovery were comparable and the difference between the two groups was even less than the difference at the end of treatment. Therefore, the statistical significance was the result of a very low variation in individual values within both groups and considered to have occurred by chance.

At the end of treatment, serum levels of T4 in treated F0 males were in the same range as in controls.

Historical control data (period 2015-2017):
Potassium (lactating females) - mean: 3.78, P5-P95:3.11-4.33 (n=186).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength in treated animals at all doses was comparable to that observed in the respective controls. Moreover, the grip strength between lactating and nulliparous females was similar in controls and females at 75 mg/kg bw/day. The variation in motor activity did not indicate a relation with treatment in main males and females. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The increase in activity towards the end of the measurement (interval 12) in the lactating (main) females is unexpected and cannot be explained. Since it was observed in all four groups at a similar activity, no toxicological significance was attached to this finding.
In recovery males as well as in recovery (nulliparous) females a difference in motor activity between high dose (75 mg/kg bw/day) and control animals was noted, with higher values for total movements and ambulations in controls, not achieving statistically significant differences. In comparison with historical control data, the motor activity in control males was higher than expected, whereas that in the high dose males was within the normal range (5). In control and high dose, recovery females the total movements as well as the ambulations were within the normal range, although rather high in controls. Since the motor activity in high dose recovery males and females was within the normal range and no treatment related effect was observed in the main males and females, the differences in motor activity between controls and high dose recovery animals were considered to have occurred by change and of no toxicological significance.

(5) Historical control data for motor activity (period 2015-2017):
Total movements: males - mean: 3341 P5-P95:1734-5284 (n=200), females 4888 P5-P95:2507-8190 (n=369)
Ambulations: males - mean: 883 P5-P95:293-1143 (n=200), females 1337 P5-P95:627-2287 (n=359)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a test item-related decrease in thymus and spleen weight (only absolute, -24% and -15%, respectively) and an increase in liver weight (only relative to body weight, +12%) in main group males treated at 75 mg/kg bw/day. In the recovery group, there was an increase in spleen weight (only relative to body weight, +13%) in males treated at 75 mg/kg bw/day.
Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a decrease in final body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were five test item-related deaths in animals, two males and three females, treated at 75 mg/kg bw/day. Four of these decedents were emaciated. Additionally, a reduced size of the thymus (in one male) and dark red foci in the glandular stomach at necropsy (in one female) were observed in single animals at necropsy. One male showed no macroscopic findings.
There were no test item-related gross observations in all other animals (surviving to scheduled necropsy) of all groups.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thyroid gland of males and females and are summarized in text table 2 and 3 (see `any other information on results incl tables`) and in the spleen, liver and bone marrow (femur and sternum) of males and are summarized in text table 4 (see `any other information on results incl tables`).
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in males starting at 25 mg/kg bw/day up to slight degree. After a 14-day treatment free period the follicular cell hypertrophy was still present at increased incidence and severity in males treated at 75 mg/kg bw/day.
Thyroid glands, an increased incidence and severity of follicular cell hypertrophy was present in females starting at 7.5 mg/kg bw/day up to moderate degree. After a 14-day treatment free period the follicular cell hypertrophy showed complete recovery.
Spleen, a decreased incidence and severity of extramedullary hematopoiesis was present in males treated at 75 mg/kg bw/day. This finding correlated with the decreased organ weight. After a 14-day treatment free period the extramedullary hematopoiesis showed complete recovery.
Spleen, an increased incidence of pigmentation (likely hemosiderin) was present in males treated at 75 mg/kg bw/day. After a 14-day treatment free period the pigmentation (likely hemosiderin) showed complete recovery.
Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day up to slight degree. This finding correlated with the increased organ weight. After a 14-day treatment free period the hepatocellular hypertrophy showed complete recovery.
Bone marrow (sternum), atrophy was present in males treated at 75 mg/kg bw/day up to slight degree. After a 14-day treatment free period the atrophy showed complete recovery.
Bone marrow (femur), atrophy was present in males treated at 75 mg/kg bw/day at minimal degree. After a 14-day treatment free period the atrophy showed complete recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
|reproduction and developmental results are included in sections 7.8.1 and 7.8.2

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
haematology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
bone marrow
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 91% and 96%). No test item was detected in the control group.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 1.2% and 1.8% in the low and high dose goups, respectively).

Note to Clinical signs, Body weight and Food consumption tables:

For Main group males, “Repro period” represents the mating phase. For Main females, “Repro period” represents the mating, post coitum and lactation phase. For Recovery group animals, the premating period and Repro period represents the treatment phase.

Note to Functional tests, Haematology, Coagulation, Clinical chemistry and Organ weights tables:

Female Main and Recovery groups were evaluated separately because of the difference in physiological status, i.e. main females were lactating and recovery females were nulliparous.

Text Table 2.
Summary Test Item-Related Microscopic Findings – Thyroid Gland Males
Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

5

5

5

5

5

5

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

1

3

2

3

2

2

      Slight           

-

-

2

1

-

2

a = Number of tissues examined from each group.

Text Table 3.

Summary Test Item-Related Microscopic Findings – Thyroid Gland Females
Scheduled Euthanasia Animals

 

Main Females

Recovery Females

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

THYROID GLANDSa

4

5

5

5

5

4

   Follicular cell hypertrophy

 

 

 

 

 

 

      Minimal

2

2

2

1

1

2

      Slight

-

2

2

2

-

-

      Moderate

-

-

   1

-

-

-

a = Number of tissues examined from each group.

Text Table 4.
Summary Test Item-Related Microscopic Findings – Spleen, Liver, Bone Marrow Males
Scheduled Euthanasia Animals

 

Main Males

Recovery males

Dose level (mg/kg/day):

0

7.5

25

75

0

75

 

 

 

 

 

 

 

SPLEENa

5

5

5

5

5

5

   Extramedullary hematopoiesis

 

 

 

 

 

 

      Minimal

4

4

4

1

4

4

      Slight

1

1

1

-

1

-

 

 

 

 

 

 

 

   Pigmentation (likely hemosiderin)

 

 

 

 

 

 

      Minimal

1

1

1

4

2

2

 

 

 

 

 

 

 

LIVERa

4

5

5

5

5

5

   Hepatocellular hypertrophy

 

 

 

 

 

 

      Minimal

-

-

-

2

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (STERNUM)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Minimal

-

-

-

1

-

-

      Slight

-

-

-

1

-

-

 

 

 

 

 

 

 

BONE MARROW (FEMUR)a

5

5

5

5

5

5

   Atrophy

 

 

 

 

 

 

      Slight

-

-

-

1

-

-

a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on observed inncreased mortality, body weight changes, effect on red blood cell formation and histopathological changes in several organs, the NOAEL for systemic (parental) toxicity in this study is 25 mg/kg bw/day.
Executive summary:

Wistar Han rats were treated with 2-(DIPHENYLPHOSPHINO) BENZOIC ACID by daily oral gavage at dose levels of 7.5, 25 and 75 mg/kg bw/day, followed by a 14-day treatment-free recovery period. The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days).

Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 51-56 days). Two females that failed to deliver (healthy) pups, i.e. a total litter loss and a non-pregnant female, were treated for 40 and 41 days, respectively. Results on reproduction and developmental toxicity are reported in sections 7.8.1. and 7.8.2

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

Salivation was noted among animals at 75 mg/kg bw/day during the treatment period. Taking into account the nature and minor severity of the salivation and its time of occurrence (i.e. after dosing), this sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

From week 3 of treatment onwards, sudden body weight loss was observed among males and females treated at 75 mg/kg bw/day. Between Days 25 and 35, three females and two males had to be sacrificed, mainly because of severe body weight loss. All these decedents showed piloerection and hunched posture and in some of them also hypothermia (one female) or lethargy (two males) was observed shortly before early sacrifice. In several animals surviving to scheduled necropsy, piloerection and/or hunched posture was also regularly observed from week 3 onwards. Additionally, lethargy was seen in two males on the day before scheduled sacrifice. In males at 75 mg/kg bw/day, the individual changes in body weight gain from week 3 of treatment onwards resulted in lower mean body weights in males at 75 mg/kg bw/day in comparison with controls. In 75 mg/kg bw/day treated main females, reduced body weight gain was observed over the first week post-coitum, and again over the first week of lactation, but no effects on body weight were observed in the non-mated, recovery females. Changes in food consumption in both sexes observed during the study were in line with the changes in body weight.

At the end of treatment, changes in haematology parameters were noted in males and (nulliparous) recovery females at 75 mg/kg bw/day only and consisted of decreased reticulocyte counts and red blood cell distribution width. In addition, increased red blood cell counts and haemoglobin and haematocrit concentrations were observed in males at the end of treatment. The combination of these changes might indicate an effect on the formation of red blood cells and, as a result, the proportion of young and old erythrocytes in the peripheral blood might have been altered. Despite the effects on red blood cells in the peripheral blood, no alterations were observed in the bone marrow after histopathological examination in these animals. In lactating females at 75 mg/kg bw/day, no treatment-related changes in haematology parameters were observed at the end of treatment.

At the end of the 14-Day recovery period, the differences in haematology parameters between the high dose animals and controls had diminished or disappeared.

At the end of treatment, changes in clinical biochemistry parameters were noted in males, lactating and (nulliparous) recovery females at 75 mg/kg bw/day. Increased potassium levels were observed in males and decreased potassium levels in lactating females. The toxicological significance of the latter was doubted, since the potassium levels obtained in controls and in 7.5 and 25 mg/kg bw/day treated females were higher than the historical control data for rats of this strain and age, whereas that in the high dose females at 75 mg/kg bw/day were within the normal range. In addition, no effect on potassium was observed in high dose recovery females at the end of treatment. Other changes at the end of treatment were observed as increased total protein in lactating females and decreased total bilirubin, urea, creatine and chloride in recovery females. Since all changes in clinical biochemistry parameters in male and female rats treated at 75 mg/kg bw/day were within the historical control range for rats of this strain and age, they were considered minimal and non-adverse in nature.

At the end of treatment, serum levels of T4 in treated F0-males were in the same range as in controls.

There were five test item-related deaths in the high dose group. No definite cause of death could be determined for these animals. For one decedent, a stomach ulceration was considered to be the cause of moribundity.

There were no test item-related macroscopic findings.

There were test item-related decreases in thymus and spleen weight (absolute) and an increase in liver (relative to body weight) in main group males treated at 75 mg/kg bw/day. There was no microscopic correlate for the organ weight changes in the thymus.

Test item-related microscopic findings were present in:

-Thyroid gland, follicular cell hypertrophy was present at slightly increased incidence and severity in females starting at 7.5 mg/kg bw/day and in males starting at 25 mg/kg bw/day. After the 14-day treatment-free period the slight increase in incidence and severity was still present in males, but had completely recovered in females.

-Spleen, extramedullary hematopoiesis was present at decreased incidence and severity in males treated at 75 mg/kg bw/day. This correlated with the decrease in organ weight. After the 14-day treatment-free period there was complete recovery. Pigmentation (likely hemosiderin) was present at increased incidence in males treated at 75 mg/kg bw/day. After the 14-day treatment-free period there was complete recovery.

-Liver, hepatocellular hypertrophy was present in males treated at 75 mg/kg bw/day. This correlated with the increase in organ weight. After the 14-day treatment-free period there was complete recovery.

-Bone marrow (femur and sternum), atrophy was present in males treated at 75 mg/kg bw/day/day. After the 14-day treatment-free period there was complete recovery.

After the 14-day treatment-free period there was an increase in spleen weight (only relative to body weight) in recovery group males at 75 mg/kg bw/day. There was no microscopic correlate to this. No signs of toxicity were observed in the parental male and female rats treated at 7.5 and 25 mg/kg bw/day.

Based on observed inncreased mortality, body weight changes, effect on red blood cell formation and histopathological changes in several organs, the NOAEL for systemic (parental) toxicity in this study is 25 mg/kg bw/day.