Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2020 - 18 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-Day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Salts of 2-ethylhexyl dihydrogen phosphate ester with C16-18-(even numbered, saturated and unsaturated)-alkylamines

Molecular formula:

C24H54[N+]O3[O-]P C26H58[N+]O3[O-]P

IUPAC Name:

Salts of 2-ethylhexyl dihydrogen phosphate ester with C16-18-(even numbered, saturated and unsaturated)-alkylamines

Test material form:

liquid

Details on test material:

Physical Description: Clear yellow liquid
Storage Conditions: At room temperature

Specific details on test material used for the study:

Physical Description: Clear yellow liquid
Storage Conditions: At room temperature

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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks old (males); 13-14 weeks old (females)
- Weight at study initiation: 280 - 339 g (males); 196 - 250 g (females)
- Fasting period before study: No, males were fasted o/n before sacrifice
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages. During the post-mating phase, males were housed in their home cage (Makrolon plastic cages) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages. During the lactation phase, females were housed in Makrolon 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), ad libitum (except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.)
- Water: Municipal water, ad libitum (During motor activity measurements, animals had no access to water for a maximum of 2 hours.)
- Contaminants: It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.
- Animal enrichment: devices for hiding in, paper and/or objects for chewing
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C (actual range)
- Humidity (%): 51 to 76% (actual range)
- Air changes (per hr): 10 or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 24 September 2020 To: 23 November 2020

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:

corn oil

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, stirred for at least 30 minutes and dosed within 24 hours after adding the vehicle to the test item or stored in the refrigerator. The dosing formulations that were removed from the refrigerator were stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
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. Adjustments were made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

- Dose volume: 5 mL/kg bw

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20244541 version 3).
Dose formulation samples were collected for analysis of concentration on Day 1 and on Day 27 (all groups). For homogeneity measurements samples of the low and the high dose groups were collected on Day 1 and Day 27.

CONCENTRATION ANALYSIS
Duplicate sets of samples 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 ANALYSIS
Duplicate sets of samples for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.

Duration of treatment / exposure:

Males: At least 28 days
Females: 50-56 days (females that delivered); 40-60 days (females which failed to deliver or had a total litter loss)

Frequency of treatment:

Once daily (high dose animals were not treated Days 10 and 11)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Animals were dosed at 500 mg/kg bw/day from Day 1 through 9. Based on significant body weight loss, animals were allowed a dosing holiday on Days 10 and 11. Dosing at 300 mg/kg bw/day was resumed on premating Day 12.

No. of animals per sex per dose:

10

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 Dose Range Finder with oral administration of the substance in rats.
In short, 3 females per group were exposed by gavage once daily to 500 or 1000 mg/kg bw/day for 14 consecutive days. The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000mg/kg bw). Mortality was checked twice daily. Clinical observations were performed at least daily from Days 1-10, at 0-15 minutes, 1 hour (± 15 minutes) and 3 hours (± 30 minutes) after dosing. Body weights were measured on Day 1 prior to dosing and on Days 5, 10 and 14. Food consumption was monitored over Days 1-5, 5-10 and 10-14. The animals dosed at 1000 mg/kg bw/day were euthanized in extremis on Day 8, due to severe clinical signs and loss of body weight (i.e. -13 to -24% within 8 days). All animals were subjected to an external, thoracic and abdominal examination at early sacrifice or on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day
- Observed for general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals
- Arena observations: Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter

FOOD CONSUMPTION measured quantitatively: Yes
- Time schedule: 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.

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was
introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: males were fasted o/n, females were not fasted
- How many animals: 5 per sex per dose
- Parameters checked in table 3 were examined.

COAGULATION
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: males were fasted o/n, females were not fasted
- How many animals: 5 per sex per dose
- Prothrombin time and activated partial thromboplastin time were analysed

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: males were fasted o/n, females were not fasted
- How many animals: 5 per sex per dose
- Parameters checked in table 5 were examined.

Thyroid hormones: Yes
- Time of blood sample collection: on the day of scheduled necropsy
- Animals fasted: males were fasted o/n, females were not fasted
- How many animals: All
- Parameters checked: Thyroxine (T4) and Thyroid stimulating hormone (TSH)

URINALYSIS: Not specified

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Week 4 of treatment (males); the last week of lactation (females)
- Dose groups that were examined: all dose groups, 5 animals per sex per dose
- Tests performed: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

ESTROUS CYCLE DETERMINATION: Yes
- Time schedule for examinations: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment, the first 14 days of treatment and during mating until evidence of copulation was observed.

GENERAL REPRODUCTION DATA
- Parameters recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day

Sacrifice and pathology:

TERMINAL PROCEDURES: Females with total litter loss were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were weighed.
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.

GROSS PATHOLOGY: All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals.

HISTOPATHOLOGY: Yes (see table 7)

Microscopic examination on all tissues was performed for all males and females in the control and high dose group. For the low and the mid dose males, also thymus, duodenum, jejunum, ileum and mesenteric lymph nodes were examined. For the low and the mid dose females the thymus, duodenum, jejunum, ileum and mesenteric lymph nodes were examined.

A detailed qualitative evaluation of the testis was conducted for all selected control and high dose animals and of males suspected to be infertile.
Testes were evaluated to assess the progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis). Any cell- or stage-specificity of testicular findings were noted.
Gross lesions were examined from all animals and correlated to microscopic findings if possible.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection was seen in females in the high dose group from Week 2 onwards. It was also observed in several females at 150 mg/kg bw/day in Weeks 5-6 and incidentally in males at 500/300 mg/kg bw/day.
Yellow staining of the anus region was noted in several females at 500/300 mg/kg bw/day. Yellow staining of the abdomen was recorded in one male at 500/300 mg/kg bw/day on a single day.
Bleeding from the nose was recorded in one female at 500/300 mg/kg bw/day. At the incidence observed, this sign was considered not to be of toxicological relevance.
Salivation was noted in males and females during daily detailed clinical observations starting at 50 mg/kg bw/day in a dose-dependent manner. Taking into account the nature and minor severity of the effects and its time of occurrence (i.e. after dosing), this sign was considered to be a physiological response rather than a sign of systemic toxicity.
Incidental findings that were noted included alopecia, hunched posture and rales. These findings 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 were considered not to be signs of toxicological relevance.
No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and two females at 500 mg/kg bw/day were euthanized in extremis on Day 9, which was considered related to treatment with the test item. For one male, piloerection and salivation were noted on Day 9. It had lost 13% of its body weight within 9 days and at necropsy watery-cloudy contents of the intestines and reduced thymus size were recorded. For one female, piloerection and yellow staining of the anus region were noted on Days 7, 8 and/or 9. It had lost 13% of its body weight within 9 days and at necropsy watery-cloudy contents of the intestines, dark red discolored kidneys and reduced thymus size were recorded.
For one female, hunched posture and piloerection were noted on Day 9. It had lost 12% of its body weight within 9 days and at necropsy watery-cloudy contents of the intestines and red discolored mesenteric lymph nodes were recorded.
One female at 150 mg/kg bw/day and one female at 500/300 mg/kg bw/day were euthanized on Lactation Day 2 and 4, respectively, as they had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to be affected by treatment with the test item starting in males at 50 mg/kg bw/day and in females at 150 mg/kg bw/day.
In males, mean body weight and mean body weight gain were decreased at 50, 150 and 500/300 mg/kg bw/day throughout the pre-mating and mating period (statistically significant at all timepoints for the high dose group and on Day 15 for the mid dose group). At the end of the treatment period, mean body weight was decreased by 5%, 8% and 15% of control mean, and body gain was -21%, -47% and -79% of control mean at 50, 150 and 500/300 mg/kg bw/day, respectively.
In females, mean body weight and mean body weight gain were decreased at 500 mg/kg bw/day between Days 1 and 9 of the premating period (Around -4% compared to controls for body weights and bodyweight gain). After a 2 day dosing holiday, mean body weight (gain) recovered to levels similar to the control on Day 12 of the premating period.

During the post coitum period and during lactation, body weights in the high dose group were lower compared to the control animals from Day 4 post coitum onwards. The difference increased and became statistically significant between post coitum Day 17 until lactation Day 1 (up to -14% post coitum Day 20, -7% Lactation Day 13 compared to controls). The body weights of the low and mid dose groups were comparable to the controls during the post coitum period and during lactation.
Body weight gain during pregnancy was affected in a dose related manner and observed during the entire period. This effect was most pronounced for the high dose group (final body weight gain on post coitum Day was -18% and -47% for the mid and the high dose group, the low dose group had the same body weight gain as controls on Day 20). Body weight gain was slightly increased during lactation period for the mid and high dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after correction for body weight was considered affected by treatment with the test item in males at 500/300 mg/kg bw/day and in females starting at 150 mg/kg bw/day.
In males, mean absolute and relative food consumption was decreased (48% and 41% of mean control, respectively) at 500 mg/kg/day over Days 1-8 of the premating period. After a 2-day dosing holiday and reduction of the dose level from 500 to 300 mg/kg bw/day, mean absolute and relative food consumption were at levels similar to the control mean over Days 1-8 of the mating period. Mean absolute food consumption was decreased (20% of control mean) over Days 8-15 of the mating period.
In females, mean absolute and relative food consumption were decreased (36% and 37% of control mean, respectively) at 500 mg/kg bw/day over Days 1-8 of the premating period (not statistically significant). After a 2-day dosing holiday and reduction of the dose level from 500 to 300 mg/kg bw/day, mean absolute and relative food consumption were at levels similar to the control mean over Days 8-15 of the premating period.
At 150 mg/kg bw/day, mean relative food consumption was decreased (14% of control mean) over Days 1-8 of the premating period.
Mean absolute and relative food consumption were decreased in females at 500/300 mg/kg bw/day over post-coitum Days 17-20 and Lactation Days 1-13 (18-35% and 8-25% of control mean).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters of treated rats were affected by treatment with the test item starting at 150 mg/kg bw/day in males and at 500/300 mg/kg bw/day in females. The results are attached below in tabular format.
The following differences were statistically significant. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses:
• Mean platelet (PLT) concentration was increased (1.29x and 1.61x of control mean) in males at 150 and 500/300 mg/kg bw/day.
• Mean of mean corpuscular volume (MCV) was decreased (0.91x of control mean) in females at 500/300 mg/kg bw/day.
The increase in mean white blood cell (WBC), neutrophil (NEUT) and monocytes (MONO) concentrations in males at 150 and 500/300 mg/kg bw/day was considered a result of one outlier in each group and was therefore considered to be of no toxicological relevance. Coagulation parameters of treated animals were considered not to have been affected by treatment with the test item up to 300 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were affected by treatment with the test item starting at 150 mg/kg bw/day in males and at 500/300 mg/kg bw/day in females. The results are attached in tabular form.
The following statistically significant changes distinguished treated from control animals. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses:
• Mean alanine aminotransferase (ALT) activity was increased (1.58x of control mean) in males at 500/300 mg/kg bw/day.
• Mean aspartate aminotransferase (AST) activity was increased (1.25x and 1.26x of control mean) in males at 150 and 500/300 mg/kg bw/day, respectively.
• Mean total bilirubin (TBIL) concentration was decreased in males and increased in females at 500/300 mg/kg bw/day (0.75x and 1.29x of control mean, respectively).
• Mean bile acid (BILEAC) concentration was increased (6.95x of control) in females at 500/300 mg/kg bw/day.
• Mean potassium concentration was increased (1.14x of control) in females at 500/300 mg/kg bw/day.
Mean glucose values in males achieving a level of statistical significance when compared to control mean were considered to have arisen as a result of slightly high control value and were therefore considered to be of no toxicological significance.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item due to the absence of a dose-related trend and/or the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Mean serum levels of total T4 were decreased (0.73x and 0.55x of control) in males at 150 and 500/300 mg/kg bw/day, respectively.
Mean serum levels of total T4 (females) and of total TSH (males and females) were considered not to be affected by treatment with the test item up to 300 mg/kg bw/day.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observation parameters in males and females were considered to be affected by treatment with the test item at 300 mg/kg bw/day. In females, mean fore limb grip strength, total number of movements and ambulations were decreased at 300 mg/kg bw/day (0.66x, 0.83x and 0.73x of control mean, respectively; not statistically significant).
In males, mean total number of movements was decreased at 300 mg/kg bw/day (0.75x of control
mean; not statistically significant).
No test item related effects were found on hearing ability, pupillary reflex and static righting reflex, mean fore limb grip strength (males) and hind limb grip strength (males and females).
In females, mean fore limb grip strength, total number of movements and ambulations were decreased at 500/300 mg/kg bw/day (0.66x, 0.83x and 0.73x of control mean, respectively; not statistically significant).
In males, mean total number of movements was decreased at 500/300 mg/kg bw/day (0.75x of control mean; not statistically significant).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-related decrease in absolute and relative weight were seen for thymus in males (absolute: -12%, -29% and -48%/ relative to body weight: -13%, -24% and -41% at 50, 150 and 300 mg/kg bw/day, resp.) and in females (absolute: -9%, -27% and -32%/ relative to body weight: -11%, -27% and -28% at 50, 150 and 300 mg/kg bw/day, resp.).
There were no other test item-related organ weight changes. Some organ weight differences (heart, liver, thyroid glands, adrenal glands, testes, epididymides, prostate gland and seminal vesicles) were statistically significant different from the control group but were considered to be the result of a test item-related effect on final body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A test item-related reduced thymus size was present in 3/8 surviving females treated at 300 mg/kg bw/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the substance were noted in the thymus, small intestines (duodenum, jejunum, ileum) and mesenteric lymph node of males and females. A summary of the test item-related microscopic findings is attached below.
In short:
Thymus: The amount of lymphocytes present in the thymus was decreased at 150 mg/kg bw/day in 1/5 males (minimal) and 2/5 females (minimal and slight) and at 300 mg/kg bw/day in 4/5 males (2 minimal, 2 slight) and 4/5 females (2 minimal, 2 moderate). This correlated to the lower thymus weight. The observed effects on thymus were considered non-adverse at the severities noted and in the absence of degenerative changes.
Duodenum: foamy macrophages in the lamina propria of the villi were present starting at 150 mg/kg/day in males up to moderate degree and in females at 500/300 mg/kg bw/day at minimal degree.
Jejunum: foamy macrophages in the lamina propria of the villi were present starting at 150 mg/kg bw/day in males and females up to moderate degree.
Ileum: foamy macrophages in the lamina propria of the villi were present starting at 150 mg/kg bw/day in males and females up to slight degree.
Mesenteric lymph node: sinus histiocytosis (foamy macrophages) was present starting at 50 mg/kg bw/day in males and females up to moderate degree. Furthermore, an increased incidence and severity of lymphangiectasis was present in females treated at 500/300 mg/kg bw/day up to slight degree. A minimal degree at a low incidence was considered to be within background. In general, the foamy macrophages and sinus histiocytosis (with foamy macrophages in sinuses) in the small intestines and mesenteric lymph node of males and females and the increased incidence and severity of lymphangiectasis in the mesenteric lymph node of females, were regarded to be a local response of the small intestines and draining mesenteric lymph node to the oral treatment with the test item.
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.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell
associations and proportions in the various stages of spermatogenesis were present.
At 500 mg/kg bw/day, in addition to the presence of foamy macrophages, there were three test item-related deaths which was considered adverse. After the reduction of the dose level to 300 mg/kg bw/day there were no premature decedents and there were no additional inflammatory processes (like granulomatous inflammation/abscesses) indicating permanent damage. In addition, there were no changes in the clinical pathology or in the hematology parameters indicating a negative impact of the foamy macrophages to normal function of the intestines and mesenteric lymph node (such as reduced uptake of nutrients). Based on this, the findings at 300 mg/kg bw/day were considered non-adverse.

Details on results:

Accuracy
The mean accuracy of lo, mid and high dose group formulations analyzed in Week 1 did not meet the criteria (i.e. 89%, 81%, 83% of the target, respectively) as well as the QC samples at concentration level of 10 mg/mL prepared on the same day. Therefore, it was decided to perform an additional analysis on the formulations prepared in Week 4.
The first test performed in Week 4 on the samples was not approved as the mean accuracy of the groups was 200±10% of the target. Therefore, the samples were re-diluted and analyzed again.
The concentrations analyzed in the formulations of the low, mid and high dose group formulations in Week 4 were in agreement with target concentrations (i.e. mean accuracies between 101% and 109%).
No test item was detected in the control group formulations.

Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 4.5%).

Results dose range finder study:
The animals dosed at 1000 mg/kg bw/day were euthanized in extremis on Day 8, due to severe clinical signs and loss of body weight (i.e. -13 to -24% within 8 days). The following clinical signs were noted in these animals: Piloerection in up to 3/3 females during Days 2-8, salivation in up to 3/3 females on Days 4-6 and 11-14, hunched posture in 3/3 females on Days 5-8, diarrhoea in 3/3 females on Days 5-8 and chromodacryorrhoea in up to 3/3 females on Days 5-8. Absolute and relative food consumption were considered markedly low between Days 1-5. Macroscopic examination revealed many grey-white foci in the forestomach and thickened wall of the duodenum, jejunum and ilium in 2/3 females (no findings were noted in the third female).
Absolute and relative liver weights were considered slightly decreased. Kidney weights were considered to be normal.
At 500 mg/kg bw/day, no mortality occurred. Clinical signs included piloerection, salivation and hunched posture on several days especially towards the end of the exposure period and were noted for all females. Moderate body weight loss (down to -4% compared to starting weight) in 1/3 females was found on Days 5 and 10, followed by recovery to starting weight between Days 10-14. Slight body weight gain was measured in 2/3 females between Days 1-10, followed by a marked increase (+7 or +9%) in body weight between Days 10-14 compared to starting weight. Absolute and relative food consumption were considered moderately low between Days 1-10. At macroscopic examination, no findings were noted. Absolute and relative liver weights were considered increased. Kidney weights were considered to be normal.
Based on the results of this dose range finder, selected dose levels for the main study were 50, 150 and 500 mg/kg bw/day.

Discussion:
One male and two females at 500 mg/kg bw/day were euthanized in extremis on Day 9 due to severe body weight loss. Their intestines contained watery-cloudy contents which may indicate an altered gastrointestinal metabolism. These deaths were considered to be adverse.

Clinical signs comprised of piloerection at 500/300 mg/kg bw/day in females was considered non-adverse as it was not accompanied by other (adverse) signs of toxicity and the incidence decreased during the course of the treatment period. In addition, yellow staining of the abdomen or anus region incidentally noted in males and females at 500/300 mg/kg bw/day was most likely related to the (excretion of) colored test item (clear yellow) and was therefore considered as non-adverse.

In males, body weight (gain) was decreased starting at 50 mg/kg bw/day throughout the treatment period. Moreover, food consumption was decreased at 500/300 mg/kg bw/day over Days 8-15 of the mating period.
In females, body weight (gain) was decreased in females at 500 mg/kg bw/day between Days 1-9 of the premating period and food consumption was decreased starting at 150 mg/kg bw/day over Days 1-8 of the premating period. Furthermore, body weight was decreased in females at 500/300 mg/kg bw/day between post‑coitum Day 17 and Lactation Day 1 and body weight gain was decreased at 150 and 500/300 mg/kg bw/day in females throughout the post-coitum period. Food consumption was decreased in females at 500/300 mg/kg bw/day between post-coitum Days 17‑20 and Lactation Days 1-13. At the magnitude of these effects, these changes in body weight (gain; in both sexes) and food consumption (in males at 500/300 mg/kg bw/day and in females starting at 150 mg/kg bw/day) were considered adverse.
The decrease in body weight (gain) in males at 50 mg/kg bw/day was considered not to be adverse due to the minimal magnitude and absence of any adverse pathological alterations.

Functional observation tests revealed no adverse changes. The lower fore limb grip strength (females), total number of movements (both sexes) and total number of ambulations (females) at the end of the treatment were considered not to represent an adverse effect on neurobehaviour. These results were not supported by clinical observations, were slight in nature (not statistically significant and within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.

Clinical pathology findings consisted of increased alanine aminotransferase, aspartate aminotransferase and platelet concentration (starting in males at 150 mg/kg bw/day), increased platelet concentration (males at 500/300 mg/kg bw/day), decreased bile acid concentration (males at 500/300 mg/kg bw/day), decreased mean corpuscular volume (females at 500/300 mg/kg bw/day), and increased bile acid and potassium concentration (females at 500/300 mg/kg bw/day). These were considered non-adverse since these changes were not associated with any adverse pathological alterations.

A marked test-item related decrease in serum levels of total T4 was noted for in F0‑males at 150 and 500/300 mg/kg bw/day.

Test item-related morphologic alterations were present in males and/or females in thymus (decreased thymus weight and lymphocyte cellularity starting in both sexes at 150 mg/kg bw/day, and macroscopically reduced thymus size in females at 500/300 mg/kg bw/day). At the severities noted and in the absence of degenerative changes, these findings were considered non-adverse.
The foamy macrophages and sinus histiocytosis (with foamy macrophages in sinuses) in the small intestines and mesenteric lymph node of males and females, and the increased incidence and severity of lymphangiectasis in the mesenteric lymph node of females were regarded to be a local response of the small intestines and draining mesenteric lymph node to the oral treatment with the test item.
At 500 mg/kg bw/day, in addition to the presence of foamy macrophages, there were three test item-related deaths which were considered adverse. After the reduction of the dose level to 300 mg/kg bw/day there were no premature decedents or additional inflammatory processes (like granulomatous inflammation/abscesses) indicating permanent damage. In addition, there were no changes in the clinical pathology parameters indicating a negative impact of the foamy macrophages to normal function of the intestines and mesenteric lymph nodes (such as reduced uptake of nutrients). Therefore, the microscopic findings in the small intestines starting at 150 mg/kg bw/day (both sexes) and in the mesenteric lymph node starting at 50 mg/kg bw/day (both sexes) were considered non-adverse.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: food consumption

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Test item-related morphologic alterations were present in males and/or females in thymus (decreased thymus weight and lymphocyte cellularity starting in both sexes at 150 mg/kg/day, and macroscopically reduced thymus size in females at 500/300 mg/kg/day). Mean serum levels of total T4 were decreased (0.73x and 0.55x of control) in males at 150 and 500/300 mg/kg/day, respectively. At the severities noted and in the absence of degenerative changes, these findings were considered non-adverse although these results indicate that the thymus is a target organ.

Applicant's summary and conclusion

Conclusions:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the substance was performed according to OECD guideline 422 and in accordance with GLP principles. Based on this study it is concluded that the (parental) No Observed Adverse Effect Level (NOAEL) is 50 mg/kg bw/day based on decreased body weight (gain) at 150 mg/kg bw/day and above.

Executive summary:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with the substance was performed according to OECD guideline 422 and in accordance with GLP principles.

The dose levels in this study were selected to be 0, 50, 150, 500 mg/kg bw/day, based on the results of the Dose Range Finder. Due to severe toxic effects observed at 500 mg/kg bw/day, the dose level was decreased to 300 mg/kg bw/day on premating Day 12. 

Chemical analyses of formulations were conducted during the study and confirmed accurate dosing.

No parental toxicity was observed up to 50 mg/kg bw/day. In males, body weight (gain) was decreased starting at 50 mg/kg bw/day throughout the treatment period (final body weights were decreased by 5%, 8% and 15% of control mean for the low, mid and high dose group, respectively). In females, body weight (gain) was decreased in females at 500 mg/kg bw/day between Days 1-9 of the premating period and food consumption was decreased starting at 150 mg/kg/day over Days 1-8 of the premating period. Furthermore, body weight was decreased in females at 500/300 mg/kg/day between post-coitum Day 17 and Lactation Day 1 and body weight gain was decreased at 150 and 500/300 mg/kg bw/day in females throughout the post-coitum period. At the magnitude of these effects, these changes in body weight (gain; in both sexes) and food consumption (in males at 500/300 mg/kg bw/day and in females starting at 150 mg/kg bw/day) were considered adverse. The decrease in body weight (gain) in males at 50 mg/kg bw/day was considered not to be adverse due to the minimal magnitude and absence of any adverse pathological alterations.
Functional observation tests revealed no adverse changes. Clinical pathology findings consisted of increased alanine aminotransferase, aspartate aminotransferase and platelet concentration (starting in males at 150 mg/kg bw/day), increased platelet concentration (males at 500/300 mg/kg bw/day), decreased bile acid concentration (males at 500/300 mg/kg bw/day), decreased mean corpuscular volume (females at 500/300 mg/kg bw/day), and increased bile acid and potassium concentration (females at 500/300 mg/kg bw/day). These were considered non-adverse since these changes were not associated with any adverse pathological alterations.
A marked test-item related decrease in serum levels of total T4 was noted for in F0-males at 150 and 500/300 mg/kg bw/day.
Test item-related morphologic alterations were present in males and/or females in thymus (decreased thymus weight and lymphocyte cellularity starting in both sexes at 150 mg/kg bw/day, and macroscopically reduced thymus size in females at 500/300 mg/kg bw/day). At the severities noted and in the absence of degenerative changes, these findings were considered non-adverse.
Foamy macrophages and sinus histiocytosis (with foamy macrophages in sinuses) in the small intestines and mesenteric lymph node of males and females, and the increased incidence and severity of lymphangiectasis in the mesenteric lymph node of females were regarded to be a local response of the small intestines and draining mesenteric lymph node to the oral treatment with the test item.
At 500 mg/kg bw/day, in addition to the presence of foamy macrophages, there were three test item-related deaths which were considered adverse. After the reduction of the dose level to 300 mg/kg/day there were no premature decedents or additional inflammatory processes (like granulomatous inflammation/abscesses) indicating permanent damage. In addition, there were no changes in the clinical pathology parameters indicating a negative impact of the foamy macrophages to normal function of the intestines and mesenteric lymph nodes (such as reduced uptake of nutrients). Therefore, the microscopic findings in the small intestines starting at 150 mg/kg bw/day (both sexes) and in the mesenteric lymph node starting at 50 mg/kg bw/day (both sexes) were considered non-adverse.

Based on this study it is concluded that the (parental) No Observed Adverse Effect Level (NOAEL) is 50 mg/kg bw/day.