[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-30 to 2020-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-30 to 2020-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Male animals: 80 – 85 days (11-12 weeks); Female animals: 80 – 85 days (11-12 weeks)
- Weight at study initiation: 346 – 433 g for male animals; 211 – 242 g for female animals
- Fasting period before study: no
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals / cage

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Contents of the standard diet for rats and mice guaranteed by the supplier.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/-3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Route of administration:

oral: gavage

Vehicle:

other: 5 % tween 20 in distilled water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 16, 40 and 90 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 1 mL (first occasion) or of 5 mL (second occasion) of each formulation (16, 40 and 90 mg/mL) and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at room temperature until analysis. Concentration of the test item in the dosing formulations varied between the range of 96.3 % and 113 % in comparison to the nominal values. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria at ca. 1 mg/mL and at ca. 200 mg/mL (97.2 and 105 % relative to nominal concentrations, respectively). Phosphoric acid, 2-Ethylhexyl ester, sodium salt proved to be stable in 5 % Tween 80 in distilled water at the intended concentrations at room temperature for five days.

Duration of treatment / exposure:

Males: 49 days
Females: 53 - 56 days

Frequency of treatment:

daily; 7 days/week

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary dose range finding study with Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt in rats as well as a non-valid previously terminated study. In this latter study, clinical signs and irritating/ corrosive effects in the stomach were detected at 600 mg/kg bw/day. Therefore, the high dose of 600 mg/kg bw/day was reduced to avoid unnecessary suffering of the animals. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

- Rationale for animal assignment: All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation.
There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups was as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups according to the actual body weight.

- Fasting period before blood sampling for clinical biochemistry: 16 h

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made on parental animals once a day, after the administration at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: paretnal males: first day of dosing, weekly thereafter and on the day of necropsy; parental females: Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum

FOOD CONSUMPTION: Yes
- Time schedule: weekly during treatment phase except during mating (pre-mating days 0, 7, 13, and post-mating days 20, 27, 34, 41 and 48 for male animals, pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals)

FOOD EFFICIENCY: no

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last treatmend, i. e. day of necropsy
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight (approx. 16 h)
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table 1 & 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last treatmend, i. e. day of necropsy
- Animals fasted: Yes, overnight (approx. 16 h)
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes
Detailed histological examinations were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.

The stomach of all animals at 80, 200 and 450 mg/kg bw/day (male and female) were histologically examined because thickening of the mucous membrane of the stomach at cardia in high dose group were detected in several animals at the necropsy.
The following organs were additionally processed and examined histologically:
- testes, epididymides, prostate and seminal vesicles with coagulating gland: no. 103 (male in control group, not mated); no. 306 (200 mg/kg bw/day, not fertilized its partner);
- ovaries, uterus with cervix, vagina: no. 123 (not mated female, control); no. 326 (female, 200 mg/kg bw/day, non-pregnant);
- stomach: no. 306 (200 mg/kg bw/day); no. 331 (female, 200 mg/kg bw/day);
- kidneys: no. 103 (male, control), no. 122 (female, control); no. 223 (female, 80 mg/kg bw/day); no. 325 and 326 (female, 200 mg/kg bw/day); no. 404 (male, 450 mg/kg bw/day);
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Other examinations:

Serum thyroid hormones
Blood samples for determination of serum levels of thyroid hormones (FT4 and TSH) were drawn in the morning hours.
Blood samples were collected from animals as follows:
- from 2-8 pups per litter on post-natal day 4 (in litters with 9 or more pups; samples were pooled by litter)
- from all dams and from 2-8 pups per litter on post-partum/post-natal day 13;
- from all parent male animals at termination on Day 49;
- from not mated and non-pregnant female animals on Day 53
Parameters as provided in table 4 were determined.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily:
Test item related clinical signs (decreased activity, narrow eye aperture, dyspnea and piloerection) were observed in male or female animals administered with 450 mg/kg bw/day with variable incidence and duration.

Nuzzling up the bedding material and salivation was also detected in male animals at 80 mg/kg bw/day and in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner regarding the onset, incidence and duration. Some animals salved also before the treatment at 200 and 450 mg/kg bw/day. Nuzzling up the bedding material and salivation appeared shortly after the administration and ceased within a short time period.
These signs might be considered to be secondary ones to the irritating effects of the test item.
Decreased activity, narrow eye aperture, enlarged and hard abdomen were observed in one male animal in the control group (1/12) from Day 45 up to the termination of the study. These signs were supported by macroscopic and histopathologic observations and were due to an individual disease.
The behavior and physical condition of all other male animals and all female animals were normal in the control group during the entire observation period (pre-mating, mating and post-mating periods in male animals; pre-mating, mating, gestation and lactation or post-mating periods in female animals).
At 80 mg/kg bw/day (low dose), salivation and nuzzling up the bedding material were noted for a single male animal (1/12) from Day 36 up to and including Day 48.
Female animals in the low dose group exhibited normal behavior and physical condition during the observation period.
At 200 mg/kg bw/day (mid dose), salivation and nuzzling up the bedding material were noted for several male animals (9/12) from Day 12 transiently or up to and including Day 48. Salivation was observed before treatment in one male animal (1/12) for two days.
In the female animals in the mid dose group, salivation and nuzzling up the bedding material were observed during the pre-mating period (6/12), during gestation (3/11) and lactation (3/11) periods.
At 450 mg/kg bw/day (high dose), salivation and nuzzling up the bedding material were noted for each male animal from Day 5 up to and including Day 48 (12/12). Besides, decreased activity (6/12), narrow eye aperture (5/12), dyspnea (4/12), piloerection (3/12) and diarrhea (1/12) were observed in male animals in the high dose group for some days. Some animal salved also before the treatment (9/12). In one male animal at 450 mg/kg bw/day (1/12), brownish hairs around the left eye was due to the overproduction of porphyrin by Harderian gland commonly seen in experimental rats.
In the female animals at 450 mg/kg bw/day, salivation and nuzzling up the bedding material were observed consistently during the pre-mating and mating (12/12), gestation (8/8) and lactation (8/8) periods or in non-pregnant and not mated female animals during the post-mating period (4/4). Salivation was observed before the treatment in some female animals at 450 mg/kg bw/day during the pre-mating period (7/12) and gestation (1/8). Abnormal gait (1/12), decreased activity (3/12), dyspnea (2/12), narrow eye aperture (2/12) and piloerection (3/12) were observed in female animals at 450 mg/kg bw/day during the pre-mating period and decreased activity (2/4), dyspnea (1/4) and piloerection (1/4) were noted for female animals during the post-mating period.

Weekly clinical observations

Adverse clinical signs related to the test item were not detected at the weekly clinical observations. Some clinical signs related to the test item (piloerection, decreased activity, abnormal gait) were sporadically (with low incidence and in some cases) detected at the detailed weekly clinical observation at 450 mg/kg bw/day. Salivation before the treatment was presumably a reflex reaction, i.e. handling of animals elicited salivation at 450 mg/kg bw/day.
Decreased activity, enlarged and hard abdomen were observed in one male animal in the control group (1/12) on Day 48.
There were no clinical signs in the other male animals in the control group and in 80 and 200 mg/kg bw/day groups.
At 450 mg/kg bw/day, salivation before the treatment (4/12), decreased activity (1/12) and piloerection (1/12) was observed in some male animals on few days at the detailed weekly observations.
Female animals in the control, 80 and 200 mg/kg bw/day groups exhibited normal behavior and physical condition at the detailed weekly observation during the entire study (pre-mating, mating, post-mating, gestation and lactation periods).
In the female animals at 450 mg/kg bw/day, salivation before the treatment (1/12 during pre-mating period, 1/8 dams during gestation period), decreased activity (2/4 non-pregnant), dyspnea (1/4 non-pregnant), piloerection (1/4 non-pregnant) and abnormal gait (1/4non-pregnant) were observed on few days at the detailed weekly observations.
Abnormal gait – observed in single female non pregnant animal at 450 mg/kg bw/day on Days 13 and 20 – was considered as an individual sign because of transient and singular occurrence.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in control, 80, 200 or 450 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was not adversely affected in male and female animals at 80, 200 and 450 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 80 and 200 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods.

The mean body weight gain was statistically significantly lower with respect to the control in the male animals at 450 mg/kg bw/day between Days 0 and 7 and in dams between gestation days 14 and 21.
The mean body weight gain was also lower with respect to the control in female animals at 450 mg/kg bw/day (no statistical significance) during the premating period.
These changes in body mean weight gain at 450 mg/kg bw/day correlated with slightly lower food consumption in the beginning of the study which might be a consequence of the local irritation effects caused by the test item. However, the changes had no influence on the overall body weight gain or on the mean body weight (male and female). Therefore, these findings were considered to be non/adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no test item related adverse effect in the mean daily food consumption of male or female animals at 80, 200 and 450 mg/kg bw/day. The mean daily food consumption was comparable in the control and test item treated male and female animals at 80 and 200 mg/kg bw/day during entire observation period.
The mean daily food consumption was slightly reduced with respect to the control in male and female animals at 450 mg/kg bw/day during the pre-mating period reaching statistical significance in male animals at 450 mg/kg bw/day during the course of the first week of the study (Day 0-7).
These changes in daily mean food intake were in accordance with body weight gain and were considered to be related to the irritating effects of the substance

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 80, 200 or 450 mg/kg bw/day.
Statistically significant difference with respect their control was detected at the higher mean percentage of neutrophil granulocytes (NEU) and at the lower mean percentage of lymphocytes (LYM) in male animals at 80 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable with the control in female animals at 80 mg/kg bw/day.
In the 200 mg/kg bw/day group, the mean platelet count (PLT) was slightly higher in male animals and the mean corpuscular hemoglobin concentration (MCHC) was slightly higher than in the control group in the female animals.
At 450 mg/kg bw/day, a higher mean white blood cell count (WBC) and higher mean percentage of neutrophil granulocytes, lower mean percentage of lymphocytes and higher mean platelet count were observed in male animals when compared with their control.
In female animals of the high dose group, statistical significance was observed at lower mean corpuscular hemoglobin concentration in comparison with their control.
These differences were considered to be toxicologically not relevant due to the minor degree (WBC, NEU, LYM, MCHC, PLT) or in the lack of dose dependency (MCHC). All values met well the ranges of the historical control data. In addition, individual renal disease in male animals no. 103 and 411 may also had influence on the WBC or LYM and NEU values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 80, 200 or 450 mg/kg bw/day (male or female).
Slight but statistically significant difference with respect to the control was detected at lower mean activity of alanine aminotransferase (ALT) in male animals at 450 mg/kg bw/day, at the lower mean sodium concentration (Na+) in male animals at 80 and 200 mg/kg bw/day and higher mean sodium concentration (Na+) in female animals at 450 mg/kg bw/day as well as at the lower mean concentration of potassium (K+) in female animals at 80 mg/kg bw/day.
These statistically significant differences with respect to their controls in male or female animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 80, 200 or 450 mg/kg bw/day groups at the end of the treatment period (on Day 47).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no adverse effects on the weights of examined organs in male or female animals at any dose level (80, 200 and 450 mg/kg bw/day).
Minor changes in the weights of the liver (relative to body or brain weights) in male animals at 80, 200 and 450 mg/kg bw/day and in the weights of prostate, seminal vesicle with coagulating gland as a whole (absolute and relative to body and brain weights), at 450 mg/kg bw/day, were considered to be of little or no biological relevance in the lack of related histopathological changes.
In the male animals, statistical significance with respect to the control was detected at the slightly higher mean spleen weight (absolute) and liver weight relative to body weight at 80 mg/kg bw/day.
At 200 mg/kg bw/day, the mean liver weights (relative to body and brain weights) exceeded the control value.
The mean weights of liver (relative to body and brain weights) were higher and the mean weight of epididymides (absolute) and mean weights of prostate, seminal vesicle with coagulating gland as a whole (absolute, relative to body and brain weights) were lower than in the control group in male animals at 450 mg/kg bw/day.
The weights of all examined organs (absolute and relative to body and brain weights) were comparable to the control in female animals at 80, 200 and 450 mg/kg bw/day.
Histopathological examinations revealed no lesions in the liver, epididymides or in prostate, seminal vesicle with coagulating gland. There were no test item-related adverse effects revealed by examination of clinical chemistry parameters indicative for an impairment of hepatic function.
Therefore, these changes were considered to be toxicologically not relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic alterations related to the effect of the test item were detected in the stomach (thickening of the mucous membrane) in a dose related manner in male and female animals at 200 and 450 mg/kg bw/day at the necropsy.
In one male animal (1/12) in the control group, macroscopic findings referring to severe individual lesion were detected as follows: extremely enlarged abdomen, smaller than normal salivary glands and thymus, enlarged lymph nodes (submandibular, mediastinal, mesenteric, abdominal), extremely enlarged and nutmeg-like patterned liver, extremely enlarged and pale spleen with light compact zone (1 cm), enlarged pituitary and sanguineous gelatinous exudate on the brain.
Pyelectasia in one or both sided kidneys (2/12) and enlarged, liquid filled kidneys (1/12, both sides) were also detected in control male animals.
In the female animals in the control group, pyelectasia (1/11 dam), moderate hydrometra (1/11 dam and 1/1 not mated female) and soft formation in the uterine horn (1/11 dam) were revealed at the necropsy.
There were no macroscopic changes in the organs or tissues of male animals of 80 mg/kg bw/day group.
In the female animals at 80 mg/kg bw/day, one side pyelectasia (1/12 dam) and moderate hydrometra (1/12 dam) were seen.
Thickened mucous membrane at cardia was observed in the stomach of one male animal at 200 mg/kg bw/day.
In the female animals at 200 mg/kg bw/day, thickened mucous membrane at cardiac part of the stomach (1/11 dam), one or both sided pyelectasia (1/11 dam and 1/1 non-pregnant female) were seen at the necropsy observations.
At 450 mg/kg bw/day, thickening of the mucous membrane at the cardiac part of the stomach (9/12 male, 4/8 dam and 3/4 non-pregnant female), one or both sided pyelectasia (3/12 male, 1/8 dam), enlarged liver (1/12 male) and enlarged, liquid filled kidney on the left side (1/12 male) were observed at the necropsy. In one dam at 450 g/kg bw/day (1/8), slight hydrometra was detected.
Thickening of the mucous membrane at the cardiac part of the stomach was considered to be related to the local effect of the test item at 200 and 450 mg/kg bw/day (male and female).
Hydrometra, related to the female sexual cycle and pyelectasia are frequent observations in untreated experimental rats of this strain. In the lack of related histopathological alterations (inflammatory or other pathological lesion) the above-mentioned findings were judged to be toxicologically not relevant.
Soft formation in the uterine horn was an individual alteration occurring in not-treated female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated reproductive organs of experimental male and female animals at 450 mg/kg bw/day.
Squamous cell hyperplasia with slight infiltration of inflammatory cells in the lamina propria in the mucous membrane of non-glandular (cardiac part of) stomach were observed in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner.
In the male animals of control and 450 mg/kg bw/day and groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each).
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.
The histology picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control and high dose groups (12/12, both).
In the female animals belonging to the high dose (450 mg/kg bw/day) treated and control groups, the ovaries, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (12/12, in control and high dose groups). The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In three female animals, showing hydrometra at the necropsy, dilatation of uterine horns was observed – 2/12 control (1/11 dam and 1/1 not mated) and 1/12 at 450 mg/kg bw/day (1/8 dam). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase). In one control dam (1/11), lipoma was seen in the uterine horn in accordance with macroscopic findings. Lipoma in the uterus is common finding in experimental rats of this strain and was observed in one control animal therefore it was without pathological significance in this study.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5, in both groups) animals.
In one control male animal (no. 105) lymphocyte leukemia (Large Granular Lymphocyte Leukemia, LGLL) was diagnosed. The neoplasm has also been referred as mononuclear cell leukemia (MCL). The diagnosis based on morphologic characteristics of cells in tissue sections. Several organs – spleen, liver, lymph nodes, kidneys, adrenal glands and brain – were involved.
Histological examination revealed in the male (12/12) and female (12/12) animals treated with 450 mg/kg bw/day focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach accompanied with slight infiltration of inflammatory cells in the lamina propria in this region of all animals.
Squamous cell hyperplasia with infiltration of inflammatory cells also occurred in one male (1/6) and in one female (1/6) animal at 200 mg/kg bw/day. No ulceration or erosion was observed in the stomach of the investigated experimental animals. The mucous membrane of the stomach in other animals at 80 and 200 mg/kg bw/day (5/5 and 5/6 male and 5/5 and 5/6 female respectively to doses) were normal at the histological investigation.
The incidences of squamous cell hyperplasia in the mucous membrane of non-glandular stomach are considered to be caused by the local irritation effects of high test item doses.
Pyelectasia in one or both sided kidneys were detected in several animal (1/7 male and 1/6 female in control group; 1/1 female at 80 mg/kg bw/day; 2/2 female at 200 mg/kg bw/day; 2/6 male and 1/5 female at 450 mg/kg bw/day) without degenerative, inflammatory or other histological (fibrotic etc.) lesion is considered as a common finding in laboratory rats without toxicological significance.
Hydronephrosis was detected in one control male (1/7, both sides) and in one male animal at 450 mg/kg bw/day (1/6, one side). This renal lesion is considered to be secondary to a mesonephric duct (Wolffian duct) defect that probably occurs during the gestation.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the small and large intestines, the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle
Test item influence on the estrous cycle was not detected at any dose level (80, 200 or 450 mg/kg bw/day).
Statistical significance with respect to the control was observed at the higher percentage of female animals with regular cycle at 80 mg/kg bw/day during the pre-mating period. There were no statistically or biologically significant differences between the control and test item treated groups (80, 200 and 450 mg/kg bw/day) in the examined parameters of estrous cycle: the mean number of cycles, the mean length of cycles, mean number of days in pro-estrous, estrous or diestrus were similar in all groups. There were no animals with prolonged estrous and the number and percentage of animals with prolonged diestrous were identical in the control and high dose groups.

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Remarks:

corresponds to 40 mg/mL

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Remarks:

corresponding to 90 mg/mL

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

Key result

Critical effects observed:

no

Table 1 Disposition of animals and outcome of pairing

Control			80 mg/kg bw/day			200 mg/kg bw/day			450 mg/kg bw/day
Animal numbers		Result of pairing	Animal numbers		Result of pairing	Animal numbers		Result of pairing	Animal numbers		Result of pairing
Male	Female		Male	Female		Male	Female		Male	Female
101	121	P/L	201	221	P/L	301	321	P/L	401	421	NP
102	122	P/L	202	222	P/L	302	321	P/L	402	422	NP
103	123	NM	203	223	P/L	303	323	P/L	403	423	P/L
104	124	P/L	204	224	P/L	304	324	P/L	404	424	P/L
105	125	P/L	205	225	P/L	305	325	P/L	405	425	P/L
106	126	P/L	206	226	P/L	306	326	NP	406	426	NP
107	127	P/L	207	227	P/L	307	327	P/L	407	427	NP
108	128	P/L	208	228	P/L	308	328	P/L	408	428	P/L
109	129	P/L	209	229	P/L	309	329	P/L	409	429	P/L
110	130	P/L	210	230	P/L	310	330	P/L	410	430	P/L
111	131	P/L	211	231	P/L	311	331	P/L	411	431	P/L
112	132	P/L	212	232	P/L	312	332	P/L	412	432	P/L

P/L = Pregnancy with live pups

NM = Not mated

NP = Non-pregnant (mated but not fertilized female)

Conclusions:

Based on the results of an OECD 422 and GLP compliant study with the test item in rat, a NOAEL of 450 mg/kg bw/d was determined for systemic toxicity and a NOAEL of 200 mg/kg bw/d (i. e. 40 mg/mL) was determined for local effects.

Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 80, 200 and 450 mg/kg bw/day compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt was administered orally (by gavage) once daily at 0 (vehicle only), 80, 200 and 450 mg/kg body weight doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 16, 40 and 90 mg/mL, corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (5 % Tween 80 in distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for five days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt concentrations in the dosing formulations varied within the range of 96.3 % and 113 % in comparison to the nominal values confirming the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 49 days). Females were additionally exposed through the gestation period and up to lactation days 12-16, i.e. up to the day before necropsy (altogether for 53-56 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-8 pups per litter (in litters with 9 or more pups) on post-natal day 4, from all dams and from 2-8 pups per litter at termination on post‑partum/post-natal day 13 and from all parent male animals and not mated, non-pregnant female animals at termination.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). These organs were processed and evaluated in non-pregnant female animal at 200 mg/kg bw/day.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in one male animal in control group with severe individual macroscopic findings in control group.

In addition, histopathological examinations were performed on the stomach of selected animals in the low and mid dose groups (on the basis of necropsy observation in high dose animals) and on organs showing macroscopic findings at the necropsy in animals in control, 80, 200 and 450 mg/kg bw/day groups.

 

There was no mortality in any groups (control, 80, 200 and 450 mg/kg bw/day).

Test item related clinical signs (decreased activity, narrow eye aperture, dyspnea or piloerection) were observed in male and female animals administered with 450 mg/kg bw/day with variable incidence and duration.

Nuzzling up the bedding material and salivation appeared in male animals at 80 mg/kg bw/day and in male and female animals at 200 and 450 mg/kg bw/day shortly after the administration and ceased within a short time period.

Some clinical signs (salivation before the treatment, piloerection, decreased activity or dyspnea) were sporadically detected at the detailed weekly clinical observation in male and female animals at 450 mg/kg bw/day.

The behavior and physical condition of the animals was normal at each dose level (80, 200 or 450 mg/kg bw/day) at the functional observations.

The body weight development was not adversely affected by the test item in male or in female animals at 80, 200 or 450 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Mean body weight gain at 450 mg/kg bw/day was reduced mainly during the pre-mating period, but had no influence on the overall body weight gain or on the mean body weight (male and female). Therefore, these findings were considered to be test item related, but non-adverse.

The mean daily food consumption was not adversely affected in male or female animals in control and at 80, 200 and 450 mg/kg bw/day during the entire study.

The slightly reduced mean food intake during the pre-mating period at 450 mg/kg bw/day correlated with the reduced body weight gain during this period. It was considered to be test item related, but non-adverse due to its minor degree.

A test item influence on the estrous cycle was not found at any dose level (80, 200 and 450 mg/kg bw/day).

There were no toxicologically relevant differences in the evaluated parameters of delivery data between the control and test item treated groups (80, 200 and 450 mg/kg bw/day). The litter size (mean number of births – total, live and viable) was slightly lowered with respect to the control in dams at 450 mg/kg bw/day. Although, the values met well the historical control ranges.

The fertility indices were reduced in male and female animals at 200 and 450 mg/kg bw/day. The individual and mean values met well the historical control ranges. In the lack of relevant changes (estrous cycle, pregnancy data, implantation sites, histopathology of sexual organs) lower mean fertility indices were considered to be insensitive factor in the determination of the NOAEL for reproductive performance

Hematological and blood coagulation investigation did not reveal adverse test item related changes in the examined parameters at 80, 200 or 450 mg/kg bw/day (male or female).

There were no test item related adverse effects on the examined clinical chemistry parameters at 80, 200 or 450 mg/kg bw/day (male or female).

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in parental males and FT4 levels in 13-day offspring. TSH levels were elevated in all test item groups in 13-day offsprings. As no dose response was seen, they were considered to be incidental and not related to test item treatment.

Macroscopic alterations related to the irritating effect of the test item were detected in the stomach (thickening of the mucous membrane) in a dose related manner in male and female animals at 200 and 450 mg/kg bw/day.

There were no adverse effects on the weights of examined organs in male or female animals at any dose level (80, 200 and 450 mg/kg bw/day). Minor changes in the weights of the liver (relative to body or brain weights) in male animals at 80, 200 and 450 mg/kg bw/day and in the weights of prostate, seminal vesicle with coagulating gland as a whole (absolute and relative to body and brain weights), at 450 mg/kg bw/day, were considered to be of little or no biological relevance in the lack of related histopathological changes.

Histopathological examinations did not reveal any toxic or other test item related lesions in the investigated reproductive organs of experimental male and female animals at 450 mg/kg bw/day.

Squamous cell hyperplasia with slight infiltration of inflammatory cells in the lamina propria in the mucous membrane of non-glandular (cardiac part of) stomach were observed in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner.

The offspring’s development was undisturbed at 80, 200 and 450 mg/kg bw/day from birth to post-natal day 13. No adverse effects on the mortality, clinical signs, body weight, anogenital distance (male and female) or nipple retention (male) were detected.

 

Under the conditions of the present study, Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt administered at 80, 200 and 450 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, parturition) in parental male and female Han:WIST rats in the absence of parental toxicity.

Test item influence on the slightly higher mean post-implantation (pre-natal) loss and lower mean birth per litter at 450 mg/kg bw/day might be presumed although values met well the historical control.

There were no clear signs of systemic toxicity in male or female animals at 80, 200 or 450 mg/kg bw/day. Transient clinical signs, minor changes in the body weight and food consumption seen at 450 mg/kg bw/day were considered as test item related, but non-adverse. Local effect was observed in the stomach at the necropsy (thickened mucous membrane at cardia) and histological examinations (squamous cell hyperplasia and inflammation) in male and female animals at 200 or 450 mg/kg bw/day. They can be ascribed to the irritating property of the test item.

There were no test item related findings in male or female animals at 80 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 80, 200 or 450 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats:  450 mg/kg bw/day

NOAEL for local toxicity of male/female rats:         200 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 450 mg/kg bw/day

NOAEL for F1 Offspring:    450 mg/kg bw/day

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (P0) animals : 14 days
- Basis for dose level selection : based on pre-test and previously terminated invalid study
- Route of administration: oral gavage

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate

Molecular formula:

C8H19O4P.2Na - C16H35O4P.Na

IUPAC Name:

Reaction mass of sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Male animals: 80 – 85 days (11-12 weeks); Female animals: 80 – 85 days (11-12 weeks)
- Weight at study initiation:
346 – 433 g for male animals; 211 – 242 g for female animals
- Fasting period before study:
no
- Housing:

Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals / cage

- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
20 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Contents of the standard diet for rats and mice guaranteed by the supplier.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/-3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 5 % tween 20 in distilled water

Details on exposure:

VEHICLE
- Concentration in vehicle:
16, 40 and 90 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1 (m:f)
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 1 mL (first occasion) or of 5 mL (second occasion) of each formulation (16, 40 and 90 mg/mL) and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at room temperature until analysis. Concentration of the test item in the dosing formulations varied between the range of 96.3 % and 113 % in comparison to the nominal values. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria at ca. 1 mg/mL and at ca. 200 mg/mL (97.2 and 105 % relative to nominal concentrations, respectively). Phosphoric acid, 2-Ethylhexyl ester, sodium salt proved to be stable in 5 % Tween 80 in distilled water at the intended concentrations at room temperature for five days.

Duration of treatment / exposure:

Males: 49 days
Females: 53 - 56 days

Frequency of treatment:

daily; 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale:
The dose levels were chosen on the basis of the results of a preliminary dose range finding study with Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt in rats as well as a non-valid previously terminated study. In this latter study, clinical signs and irritating/ corrosive effects in the stomach were detected at 600 mg/kg bw/day. Therefore, the high dose of 600 mg/kg bw/day was reduced to avoid unnecessary suffering of the animals. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

- Rationale for animal assignment:
All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation.
There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups was as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups according to the actual body weight.

- Fasting period before blood sampling for clinical biochemistry:
16 h

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made on parental animals once a day, after the administration at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: paretnal males: first day of dosing, weekly thereafter and on the day of necropsy; parental females: Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum

FOOD CONSUMPTION: Yes
- Time schedule: weekly during treatment phase except during mating (pre-mating days 0, 7, 13, and post-mating days 20, 27, 34, 41 and 48 for male animals, pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals)

FOOD EFFICIENCY: no

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were also prepared from each female animal on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 14 days of age

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

Statistics:

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Reproductive indices:

Parental Males:
- Male copulatory index: Number of males with confirmed mating / total number of males cohabited x 100
- Male fertility index: Number of males impregnating for a female / total number of males with confirmed mating x 100

Parental Females:
- Female copulatory index: Number of females with confirmed mating / total number of females cohabited x 100
- Female fertility index: Number of pregnant female / number of spermpositive females x 100
- Gestation index: Number of females with liveborn pubs / number of pregnant females x 100
- Post-implantation mortality: Number of implantations - Number of liveborns / Number of implantations x 100

Offspring viability indices:

- Survival Index of pups on postnatal day 13: Number of live pups on postnatal days 13 / Number of live borns x 100
- Sex ratio % (on postnatal days 0 and 13): Number of pups examined - Number of males (females) / Number of pups examined x 100
- postnatal mortality: Number of liveborns - Number of live pups on PND 13 / Number of liveborns x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily:
Test item related clinical signs (decreased activity, narrow eye aperture, dyspnea and piloerection) were observed in male or female animals administered with 450 mg/kg bw/day with variable incidence and duration.

Nuzzling up the bedding material and salivation was also detected in male animals at 80 mg/kg bw/day and in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner regarding the onset, incidence and duration. Some animals salved also before the treatment at 200 and 450 mg/kg bw/day. Nuzzling up the bedding material and salivation appeared shortly after the administration and ceased within a short time period.
These signs might be considered to be secondary ones to the irritating effects of the test item.
Decreased activity, narrow eye aperture, enlarged and hard abdomen were observed in one male animal in the control group (1/12) from Day 45 up to the termination of the study. These signs were supported by macroscopic and histopathologic observations and were due to an individual disease.
The behavior and physical condition of all other male animals and all female animals were normal in the control group during the entire observation period (pre-mating, mating and post-mating periods in male animals; pre-mating, mating, gestation and lactation or post-mating periods in female animals).
At 80 mg/kg bw/day (low dose), salivation and nuzzling up the bedding material were noted for a single male animal (1/12) from Day 36 up to and including Day 48.
Female animals in the low dose group exhibited normal behavior and physical condition during the observation period.
At 200 mg/kg bw/day (mid dose), salivation and nuzzling up the bedding material were noted for several male animals (9/12) from Day 12 transiently or up to and including Day 48. Salivation was observed before treatment in one male animal (1/12) for two days.
In the female animals in the mid dose group, salivation and nuzzling up the bedding material were observed during the pre-mating period (6/12), during gestation (3/11) and lactation (3/11) periods.
At 450 mg/kg bw/day (high dose), salivation and nuzzling up the bedding material were noted for each male animal from Day 5 up to and including Day 48 (12/12). Besides, decreased activity (6/12), narrow eye aperture (5/12), dyspnea (4/12), piloerection (3/12) and diarrhea (1/12) were observed in male animals in the high dose group for some days. Some animal salved also before the treatment (9/12). In one male animal at 450 mg/kg bw/day (1/12), brownish hairs around the left eye was due to the overproduction of porphyrin by Harderian gland commonly seen in experimental rats.
In the female animals at 450 mg/kg bw/day, salivation and nuzzling up the bedding material were observed consistently during the pre-mating and mating (12/12), gestation (8/8) and lactation (8/8) periods or in non-pregnant and not mated female animals during the post-mating period (4/4). Salivation was observed before the treatment in some female animals at 450 mg/kg bw/day during the pre-mating period (7/12) and gestation (1/8). Abnormal gait (1/12), decreased activity (3/12), dyspnea (2/12), narrow eye aperture (2/12) and piloerection (3/12) were observed in female animals at 450 mg/kg bw/day during the pre-mating period and decreased activity (2/4), dyspnea (1/4) and piloerection (1/4) were noted for female animals during the post-mating period.

Weekly clinical observations

Adverse clinical signs related to the test item were not detected at the weekly clinical observations. Some clinical signs related to the test item (piloerection, decreased activity, abnormal gait) were sporadically (with low incidence and in some cases) detected at the detailed weekly clinical observation at 450 mg/kg bw/day. Salivation before the treatment was presumably a reflex reaction, i.e. handling of animals elicited salivation at 450 mg/kg bw/day.
Decreased activity, enlarged and hard abdomen were observed in one male animal in the control group (1/12) on Day 48.
There were no clinical signs in the other male animals in the control group and in 80 and 200 mg/kg bw/day groups.
At 450 mg/kg bw/day, salivation before the treatment (4/12), decreased activity (1/12) and piloerection (1/12) was observed in some male animals on few days at the detailed weekly observations.
Female animals in the control, 80 and 200 mg/kg bw/day groups exhibited normal behavior and physical condition at the detailed weekly observation during the entire study (pre-mating, mating, post-mating, gestation and lactation periods).
In the female animals at 450 mg/kg bw/day, salivation before the treatment (1/12 during pre-mating period, 1/8 dams during gestation period), decreased activity (2/4 non-pregnant), dyspnea (1/4 non-pregnant), piloerection (1/4 non-pregnant) and abnormal gait (1/4non-pregnant) were observed on few days at the detailed weekly observations.
Abnormal gait – observed in single female non pregnant animal at 450 mg/kg bw/day on Days 13 and 20 – was considered as an individual sign because of transient and singular occurrence.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no mortality in control, 80, 200 or 450 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was not adversely affected in male and female animals at 80, 200 and 450 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 80 and 200 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods.

The mean body weight gain was statistically significantly lower with respect to the control in the male animals at 450 mg/kg bw/day between Days 0 and 7 and in dams between gestation days 14 and 21.
The mean body weight gain was also lower with respect to the control in female animals at 450 mg/kg bw/day (no statistical significance) during the premating period.
These changes in body mean weight gain at 450 mg/kg bw/day correlated with slightly lower food consumption in the beginning of the study which might be a consequence of the local irritation effects caused by the test item. However, the changes had no influence on the overall body weight gain or on the mean body weight (male and female). Therefore, these findings were considered to be non/adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no test item related adverse effect in the mean daily food consumption of male or female animals at 80, 200 and 450 mg/kg bw/day. The mean daily food consumption was comparable in the control and test item treated male and female animals at 80 and 200 mg/kg bw/day during entire observation period.
The mean daily food consumption was slightly reduced with respect to the control in male and female animals at 450 mg/kg bw/day during the pre-mating period reaching statistical significance in male animals at 450 mg/kg bw/day during the course of the first week of the study (Day 0-7).
These changes in daily mean food intake were in accordance with body weight gain and were considered to be related to the irritating effects of the substance

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 80, 200 or 450 mg/kg bw/day.
Statistically significant difference with respect their control was detected at the higher mean percentage of neutrophil granulocytes (NEU) and at the lower mean percentage of lymphocytes (LYM) in male animals at 80 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable with the control in female animals at 80 mg/kg bw/day.
In the 200 mg/kg bw/day group, the mean platelet count (PLT) was slightly higher in male animals and the mean corpuscular hemoglobin concentration (MCHC) was slightly higher than in the control group in the female animals.
At 450 mg/kg bw/day, a higher mean white blood cell count (WBC) and higher mean percentage of neutrophil granulocytes, lower mean percentage of lymphocytes and higher mean platelet count were observed in male animals when compared with their control.
In female animals of the high dose group, statistical significance was observed at lower mean corpuscular hemoglobin concentration in comparison with their control.
These differences were considered to be toxicologically not relevant due to the minor degree (WBC, NEU, LYM, MCHC, PLT) or in the lack of dose dependency (MCHC). All values met well the ranges of the historical control data. In addition, individual renal disease in male animals no. 103 and 411 may also had influence on the WBC or LYM and NEU values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 80, 200 or 450 mg/kg bw/day (male or female).
Slight but statistically significant difference with respect to the control was detected at lower mean activity of alanine aminotransferase (ALT) in male animals at 450 mg/kg bw/day, at the lower mean sodium concentration (Na+) in male animals at 80 and 200 mg/kg bw/day and higher mean sodium concentration (Na+) in female animals at 450 mg/kg bw/day as well as at the lower mean concentration of potassium (K+) in female animals at 80 mg/kg bw/day.
These statistically significant differences with respect to their controls in male or female animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 80, 200 or 450 mg/kg bw/day groups at the end of the treatment period (on Day 47).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated reproductive organs of experimental male and female animals at 450 mg/kg bw/day.
Squamous cell hyperplasia with slight infiltration of inflammatory cells in the lamina propria in the mucous membrane of non-glandular (cardiac part of) stomach were observed in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner.
In the male animals of control and 450 mg/kg bw/day and groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each).
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.
The histology picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control and high dose groups (12/12, both).
In the female animals belonging to the high dose (450 mg/kg bw/day) treated and control groups, the ovaries, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (12/12, in control and high dose groups). The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In three female animals, showing hydrometra at the necropsy, dilatation of uterine horns was observed – 2/12 control (1/11 dam and 1/1 not mated) and 1/12 at 450 mg/kg bw/day (1/8 dam). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase). In one control dam (1/11), lipoma was seen in the uterine horn in accordance with macroscopic findings. Lipoma in the uterus is common finding in experimental rats of this strain and was observed in one control animal therefore it was without pathological significance in this study.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5, in both groups) animals.
In one control male animal (no. 105) lymphocyte leukemia (Large Granular Lymphocyte Leukemia, LGLL) was diagnosed. The neoplasm has also been referred as mononuclear cell leukemia (MCL). The diagnosis based on morphologic characteristics of cells in tissue sections. Several organs – spleen, liver, lymph nodes, kidneys, adrenal glands and brain – were involved.
Histological examination revealed in the male (12/12) and female (12/12) animals treated with 450 mg/kg bw/day focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach accompanied with slight infiltration of inflammatory cells in the lamina propria in this region of all animals.
Squamous cell hyperplasia with infiltration of inflammatory cells also occurred in one male (1/6) and in one female (1/6) animal at 200 mg/kg bw/day. No ulceration or erosion was observed in the stomach of the investigated experimental animals. The mucous membrane of the stomach in other animals at 80 and 200 mg/kg bw/day (5/5 and 5/6 male and 5/5 and 5/6 female respectively to doses) were normal at the histological investigation.
The incidences of squamous cell hyperplasia in the mucous membrane of non-glandular stomach are considered to be caused by the local irritation effects of high test item doses.
Pyelectasia in one or both sided kidneys were detected in several animal (1/7 male and 1/6 female in control group; 1/1 female at 80 mg/kg bw/day; 2/2 female at 200 mg/kg bw/day; 2/6 male and 1/5 female at 450 mg/kg bw/day) without degenerative, inflammatory or other histological (fibrotic etc.) lesion is considered as a common finding in laboratory rats without toxicological significance.
Hydronephrosis was detected in one control male (1/7, both sides) and in one male animal at 450 mg/kg bw/day (1/6, one side). This renal lesion is considered to be secondary to a mesonephric duct (Wolffian duct) defect that probably occurs during the gestation.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the small and large intestines, the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Test item influence on the estrous cycle was not detected at any dose level (80, 200 or 450 mg/kg bw/day).
Statistical significance with respect to the control was observed at the higher percentage of female animals with regular cycle at 80 mg/kg bw/day during the pre-mating period. There were no statistically or biologically significant differences between the control and test item treated groups (80, 200 and 450 mg/kg bw/day) in the examined parameters of estrous cycle: the mean number of cycles, the mean length of cycles, mean number of days in pro-estrous, estrous or diestrus were similar in all groups. There were no animals with prolonged estrous and the number and percentage of animals with prolonged diestrous were identical in the control and high dose groups

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not adversely affected by the test item at 80, 200 or 450 mg/kg bw/day in male or female animals.
The copulatory index was slightly lower in the control group than in the test item treated groups in male and female animals.
Statistically significant difference with respect to the control was observed at the slightly lower percentage of fertile male animals and pregnant female animal, i.e. lower fertility indices at 200 and 450 mg/kg bw/day in male and female animals. The individual and mean values met well the historical control ranges. In the lack of relevant changes (estrous cycle, pregnancy data, implantation sites, histopathology of sexual organs) lower mean fertility indices were considered to be insensitive factor in the determination of the NOAEL.
There was no difference between the control and test item treated groups in the gestation index (female animals), in the mean pre-coital interval and mean conceiving days.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

3

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold and not suckled) was slightly higher in the control and low dose group than in 200 and 450 mg/kg bw/day groups.
The clinical signs in offspring were observed as follows:
- Cold body temperature: 18 % in control, 10 % in 80 mg/kg bw/day groups:
- No milk in the stomach: 2 % in control group;
- Hematoma: 1 % in control, 1 % in 80 mg/kg bw/day and 2 % in 200 mg/kg bw/day groups;
- Thin hairs: 1 %at 80 mg/kg bw/day;
These signs are common in offspring of this strain and occurred independently from doses in this study.
(Appendices 14.1 and 14.2)

9.19 Anogenital distance and nipple retention

The anogenital distances (absolute and normalized in male or female offspring) or nipple retention (male) were not adversely affected by the test item treatment at 80, 200 and 450 mg/kg bw/day.
Statistical significance was observed at the shorter mean absolute anogenital distance with respect to their control in male offspring at 80 mg/kg bw/day. The normalized anogenital distance of these pups was comparable with their control.
The absolute and normalized anogenital distances were similar to the control in female pups at 80 mg/kg bw/day and in male and female offspring at 200 and 450 mg/kg bw/day.
In the lack of dose relevance and the minor degree of difference, this finding was considered to be toxicologically not relevant.
Nipples/areoles were not visible in any of the examined male offspring in the control or 80, 200 or 450 mg/kg bw/day groups on postnatal day 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number and percentage of dead offspring were similar in the control and test item treated groups and met well the historical control.
The survival indices were similar in the control and test item treated (80, 200 or 450 mg/kg bw/day) groups.
Since there was a statistically significant lower mean number of birth at 450 mg/kg bw/day, the number of liveborn offspring and viable pups on post-natal day 0 and 4 were also statistically significantly lower in comparison to the control group numbers. The number of viable pups did not differ between post-natal day 0 and 4. Thus, the lower mean numbers were not related to a higher post-natal mortality.
The mean number of pups euthanized on postnatal day 4 was lower than in the control group at 200 and 450 mg/kg bw/day to follow the requirements of the test guideline OECD422 (elimination of pups to yield 4 pups per sex per litter).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of the offspring was undisturbed between post-natal days 0 and 13.
Slight differences in the litter weight with respect to the control was originated from the lower number of pups per litter at 450 mg/kg bw/day as there were no differences in the mean body weight of pups between the control and 450 mg/kg bw/day.
Statistical significance was observed at the lower mean litter weight comparing to the control at 200 and 450 mg/kg bw/day on the day of birth and at 450 mg/kg bw/day on pos-natal day 4.
The mean litter weight gain was slightly lower than in the control group between pots-natal days 4 and 13.
The mean body weight of pups was comparable in the control and 80 and 200 mg/kg bw/day group during the observation period.
At 450 mg/kg bw/day, statistical significance with respect to the control was detected at the slightly lower mean body weight of offspring on post-natal day 13 and at the lower mean body weight gain of offspring between post-natal days 0 and 13.
The mean body weight was lower than in the control both in male and female pups at 80 mg/kg bw/day and in female pups at 200 mg/kg bw/day on post-natal day 4, calculating separately by genders.
These differences with respect to the control were minor and were considered to be toxicologically not relevant.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related macroscopic findings in pups subjected to early necropsy or terminally.
Autolyzed visceral organs were seen in dead pup at 80 mg/kg bw/day (1/1) and hematoma on the chest in one pup (1/2) at 450 mg/kg bw/day.
All pups euthanized terminally (22/22, 24/24, 21/21 and 16/16, respectively to groups control 80, 200 and 450 mg/kg bw/day) were without macroscopic findings at the necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution:
There were no test item related differences between the control and test item treated groups in the percentage of genders on postnatal days 0, 4 or 13.
The mean number of male pups per litter was slightly lower than in the control group at 450 mg/kg bw/day at birth (post-natal day 0) and on post-natal day 4.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at highest dose tested, inducing maternal toxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of an OECD 422  and GLP compliant study with the test item in rat, the following NOAELs were determined:
NOAELsystemic: 450 mg/kg bw/d
NOAELlocal: 200 mg/kg bw/d
NOAELreproduction: 450 mg/kg bw/d
NOAELdevelopment: 450 mg/kg bw/d

Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 80, 200 and 450 mg/kg bw/day compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt was administered orally (by gavage) once daily at 0 (vehicle only), 80, 200 and 450 mg/kg body weight doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 16, 40 and 90 mg/mL, corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (5 % Tween 80 in distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for five days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt concentrations in the dosing formulations varied within the range of 96.3 % and 113 % in comparison to the nominal values confirming the proper preparation of the dosing formulations. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 49 days). Females were additionally exposed through the gestation period and up to lactation days 12-16, i.e. up to the day before necropsy (altogether for 53-56 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-8 pups per litter (in litters with 9 or more pups) on post-natal day 4, from all dams and from 2-8 pups per litter at termination on post‑partum/post-natal day 13 and from all parent male animals and not mated, non-pregnant female animals at termination.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). These organs were processed and evaluated in non-pregnant female animal at 200 mg/kg bw/day.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in one male animal in control group with severe individual macroscopic findings in control group.

In addition, histopathological examinations were performed on the stomach of selected animals in the low and mid dose groups (on the basis of necropsy observation in high dose animals) and on organs showing macroscopic findings at the necropsy in animals in control, 80, 200 and 450 mg/kg bw/day groups.

 

There was no mortality in any groups (control, 80, 200 and 450 mg/kg bw/day).

Test item related clinical signs (decreased activity, narrow eye aperture, dyspnea or piloerection) were observed in male and female animals administered with 450 mg/kg bw/day with variable incidence and duration.

Nuzzling up the bedding material and salivation appeared in male animals at 80 mg/kg bw/day and in male and female animals at 200 and 450 mg/kg bw/day shortly after the administration and ceased within a short time period.

Some clinical signs (salivation before the treatment, piloerection, decreased activity or dyspnea) were sporadically detected at the detailed weekly clinical observation in male and female animals at 450 mg/kg bw/day.

The behavior and physical condition of the animals was normal at each dose level (80, 200 or 450 mg/kg bw/day) at the functional observations.

The body weight development was not adversely affected by the test item in male or in female animals at 80, 200 or 450 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Mean body weight gain at 450 mg/kg bw/day was reduced mainly during the pre-mating period, but had no influence on the overall body weight gain or on the mean body weight (male and female). Therefore, these findings were considered to be test item related, but non-adverse.

The mean daily food consumption was not adversely affected in male or female animals in control and at 80, 200 and 450 mg/kg bw/day during the entire study.

The slightly reduced mean food intake during the pre-mating period at 450 mg/kg bw/day correlated with the reduced body weight gain during this period. It was considered to be test item related, but non-adverse due to its minor degree.

A test item influence on the estrous cycle was not found at any dose level (80, 200 and 450 mg/kg bw/day).

There were no toxicologically relevant differences in the evaluated parameters of delivery data between the control and test item treated groups (80, 200 and 450 mg/kg bw/day). The litter size (mean number of births – total, live and viable) was slightly lowered with respect to the control in dams at 450 mg/kg bw/day. Although, the values met well the historical control ranges.

The fertility indices were reduced in male and female animals at 200 and 450 mg/kg bw/day. The individual and mean values met well the historical control ranges. In the lack of relevant changes (estrous cycle, pregnancy data, implantation sites, histopathology of sexual organs) lower mean fertility indices were considered to be insensitive factor in the determination of the NOAEL for reproductive performance

Hematological and blood coagulation investigation did not reveal adverse test item related changes in the examined parameters at 80, 200 or 450 mg/kg bw/day (male or female).

There were no test item related adverse effects on the examined clinical chemistry parameters at 80, 200 or 450 mg/kg bw/day (male or female).

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose in parental males and FT4 levels in 13-day offspring. TSH levels were elevated in all test item groups in 13-day offsprings. As no dose response was seen, they were considered to be incidental and not related to test item treatment.

Macroscopic alterations related to the irritating effect of the test item were detected in the stomach (thickening of the mucous membrane) in a dose related manner in male and female animals at 200 and 450 mg/kg bw/day.

There were no adverse effects on the weights of examined organs in male or female animals at any dose level (80, 200 and 450 mg/kg bw/day). Minor changes in the weights of the liver (relative to body or brain weights) in male animals at 80, 200 and 450 mg/kg bw/day and in the weights of prostate, seminal vesicle with coagulating gland as a whole (absolute and relative to body and brain weights), at 450 mg/kg bw/day, were considered to be of little or no biological relevance in the lack of related histopathological changes.

Histopathological examinations did not reveal any toxic or other test item related lesions in the investigated reproductive organs of experimental male and female animals at 450 mg/kg bw/day.

Squamous cell hyperplasia with slight infiltration of inflammatory cells in the lamina propria in the mucous membrane of non-glandular (cardiac part of) stomach were observed in male and female animals at 200 and 450 mg/kg bw/day in a dose related manner.

The offspring’s development was undisturbed at 80, 200 and 450 mg/kg bw/day from birth to post-natal day 13. No adverse effects on the mortality, clinical signs, body weight, anogenital distance (male and female) or nipple retention (male) were detected.

 

Under the conditions of the present study, Phosphoric Acid, 2-ethylhexyl Ester, Sodium Salt administered at 80, 200 and 450 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, parturition) in parental male and female Han:WIST rats in the absence of parental toxicity.

Test item influence on the slightly higher mean post-implantation (pre-natal) loss and lower mean birth per litter at 450 mg/kg bw/day might be presumed although values met well the historical control.

There were no clear signs of systemic toxicity in male or female animals at 80, 200 or 450 mg/kg bw/day. Transient clinical signs, minor changes in the body weight and food consumption seen at 450 mg/kg bw/day were considered as test item related, but non-adverse. Local effect was observed in the stomach at the necropsy (thickened mucous membrane at cardia) and histological examinations (squamous cell hyperplasia and inflammation) in male and female animals at 200 or 450 mg/kg bw/day. They can be ascribed to the irritating property of the test item.

There were no test item related findings in male or female animals at 80 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 80, 200 or 450 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats:  450 mg/kg bw/day

NOAEL for local toxicity of male/female rats:         200 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 450 mg/kg bw/day

NOAEL for F1 Offspring:    450 mg/kg bw/day