Trihexyl phosphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct 2018 – 09 Jan 2019

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:

31 Oct 2018 – 09 Jan 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

outbreed

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Pushchino, Russia
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 348 ± 27 g (males) and 213 ± 14 g (females)
- Housing: Animals were housed in groups of 2/sex in polycarbonate cages type IV (595 x 380 x 200 mm) until mating. During the mating period, males were housed alone and cohabited with females in the home cage of the male. After mating, females were housed individually in these cages. Males and females of the satellite group (not mated) were housed 2-3 animals per cage. Commercial autoclaved woodchip bedding was used.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300, Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water: filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 30 - 63
- Air changes (per hr): at least 12 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Aug 2018 To: 09 Jan 2019

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required volumes of the test item (taking into account the purity and the density of the liquid) were mixed with the calculated volume of vehicle (water) and stirred to create a homogeneous water emulsion. Dosing formulations were prepared every three days, aliquoted to the required volumes of days of administration and stored at room temperature in tightly closed glass jars in the fume hood. On the day of dosing, the aliquot of each formulation was mixed thoroughly and stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 5, 20 and 80 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations were analysed for homogeneity, stability and concentration. Stability of the test item in the vehicle prepared at concentrations of 5, 20 and 80 mg/mL was confirmed following 3 days of storage at room temperature during method validation study. Additional data on the stability of dose formulations were obtained by analyzing the medium strata of all dose formulations after their 2-day storage and resuspension at the beginning of in-life phase of the study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a LC/MS validated method at the beginning, in the middle and at the end of in-life phase. In addition, homogeneity analysis was performed at the beginning of the study after 2 days of dose storage after resuspension.
For stability, the mean concentration of formulation samples of 5 and 80 mg/mL after 3 days of storage and resuspension should be within the acceptable range of the target concentration (85 – 115% with RSD < 10%), and within the range 85-115% of the time zero point.
Stability of the test substance formulations was confirmed after three days of storage at room temperature and after 2-day storage incl. resuspension, except for dose group 2 (20 mg/mL) which exceeded the acceptable range (120.5%) presumably due to problems of homogenization and sample preparing.
For dose formulations 5, 20 and 80 mg/mL all analysed concentrations were within the acceptable range of 85 – 115% of the target concentration for all dates of analysis, excluding formulation 20 mg/mL on date of 27 Nov 2018, for which the actual concentration was 81.0% of the target level.
The samples were considered homogeneous since the relative standard deviation (RSD) for the mean concentration was ≤ 10% at a concentration within the acceptable limits (85 – 115% of target concentration) with the following exceptions: The RSD value exceeded 10% on date of 27 Nov 2018 for 20 mg/mL (10.2%) and 80 mg/mL (12%) with a mean concentration for 20 mg/mL formulation less than the acceptable limit (79.6%). The homogeneity data of the formulations after 2-day storage and re-mixing were outside the acceptable range for the formulation of 5 mg/mL (RSD 10.9%) and 20 mg/mL (RSD 10.2% and mean concentration 117.8% of target). However, the described outputs from the acceptable range were slight, and the deviations are considered to be mild. In general, it can be concluded that dose formulations homogeneity was acceptable, not affecting the interpretation of calculated dosages administered to animals.

Duration of treatment / exposure:

Males: Treatment for 28 days, beginning 14 days prior to mating, throughout the mating period until necropsy
Females: Females which delivered were treated for 49 – 56 days, beginning 14 days prior to mating until lactation day 13. Females which failed to deliver/ with no evidence of mating were treated for 55 days.
Animals of the recovery group were not mated and dosed for 28 days (males) or 55 days (females) with following two weeks recovery period.

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main group)
5 (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Available toxicity data of structurally similar analogue substances revealed that doses of the test item used in the study on repeated dose toxicity can be in the range of 25 to 400 mg/kg bw/day. A pilot dose-range finding study with the test item was performed and confirmed that a dose of 400 mg/kg bw/day will be tolerated without causing death or obvious suffering. Test item doses of 25 and 400 mg/kg bw/day were administered daily for 21 days to each three male and three female Sprague Dawley rats. No mortalities and no adverse clinical signs were observed, except for excessive salivation in one female treated at 400 mg/kg bw/day. No effects on food consumption and body weight gain was noted and there were no gross macroscopic findings at necropsy. Increased liver weights were observed in males at 25 and 400 mg/kg bw/day and in females at 400 mg/kg bw/day and assumed to be caused by hepatocellular hypertrophy. Based on these results and based on the published data of analogues toxicity, doses of 25, 100 and 400 mg/kg bw/day were chosen for the current study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/morbidity
- Cage side observations included: All animals were observed for general condition and clinical signs of toxicity 5 - 20 min following dose administration. Females expected to deliver were observed twice daily for dystocia and other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and on a weekly basis throughout the study
- Observations included: chromodacryorrhea, palpebral aperture, hunching, piloerection, respiration, alertness, reactivity to handling, vocalization, mucous membranes, locomotion/hypokinesia, gait and stereotypes/bizzares

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on Day 1 prior to dosing and weekly thereafter. During pregnancy, mated females were weighed on Days 0, 7, 14, 20 and within 24 h of parturition and on post-natal days (PND) 2, 4 and 13.

FOOD CONSUMPTION: Yes
- Food consumption was measured prior to dosing and weekly until cohabition. Food consumption was not recorded during mating. After mating, food consumption of females was measured on gestation Days 0-1, 6-7, 13-14 and 19-20 and on lactation Days 1-2, 4-5 and 12-13.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once prior to start of treatment and on a weekly basis throughout the study.
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all satellite animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia. Tail-tip amputation was performed for assessing the level of glucose.
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all satellite animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: glucose, protein, bilirubin, urobilinogen, pH value, specific gravity, blood cells, ketones, nitrites, leukocytes, creatinine

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Day 28 of dose administration (males) and on lactation Day 13 (females)
- How many animals: 6/sex randomly selected
- Dose groups that were examined: all dose groups
- Battery of functions tested: home cage / handling / open field / sensory activity / grip strength / locomotor activity

IMMUNOLOGY: No

OTHER:
THYROID HORMONE ANALYSIS
- Time schedule for collection of blood: on the day of scheduled necropsy from the inferior vena cava; for satellite animals following two weeks post-treatment as part of euthanasia
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: Yes
- How many animals: 6/sex randomly selected of each main group; all recovery animals
- Parameters checked: thyroxine (T4)

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals were euthanized by anesthesia (Zoletil® / Xyla®) following completion of the mating period and 28 days of dose administration.
- Female animals: All surviving dams were euthanized on lactation Day 14 after litters have been euthanized on post-natal Day (PND) 13. Females which have not delivered were euthanized after 55 days of dosing. All females were euthanized by anesthesia (Zoletil® / Xyla®).
- Satellite animals: All satellite animals were subjected to euthanasia at the end of two weeks post-treatment period. All animals were euthanized by anesthesia (Zoletil® / Xyla®).

GROSS NECROPSY
- Gross necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: adrenals, brain, cowper’s glands, epididymides, heart, glans penis, kidneys, levator ani and bulbocavernosus muscles complex, liver, ovaries, pituitary, prostate, seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid and uterus. The tissues indicated in Table No. 3 were prepared for light microscopic examination from six randomly selected adult males and females in the control and high-dose groups. Liver, kidneys, spleen, stomach, testes, epididymides, and seminal vesicles with coagulating glands were examined in the low- and mid dose groups as well as in satellite subgroup animals. Microscopic examination of thyroid glands was done for all adult males and femlaes in all groups including satellite subgroups, and one male and female of day 13 pup from each litter in all groups.

Statistics:

All statistical tests were performed separately for each sex and compared the treated groups to the control group.
- Multi-factor analysis of variance ANOVA-2, followed by the Duncan test: mean body weights, body weight gain and food consumption data
- Parametric one-way analysis of variance (ANOVA), followed by Dunnett's test in case of significance (p < 0.05): clinical pathology values and functional observational battery (FOB) data, organ weights (absolute and relative to body weights and relative to brain weights)
- T-test: clinical pathology values and FOB values of satellite animals, comparison of organ weights
- Fisher’s exact test: FOB parameters which yield scalar or descriptive data, histopathological findings
- Kruskal-Wallis nonparametric ANOVA test with Dunn’s test: gamma-glutamyltransferase data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: piloerection, reactivity to handling and contraction of whisker pad (1/12 males); nasal red-brown discharge/crust and rales (1/12 females): non-adverse
100 mg/kg bw/day: nasal red-brown discharge/crust (1/12 males), transient effect; piloerection (1/12 males): non-adverse
400 mg/kg bw/day: excessive salivation 15 min after administration (4/17 males, 13/17 females); chromodacryorrhea (3/17 females); piloerection and reactivity to handling (2/17 females); nasal red-brown discharge/crust (1/17 males, 2/17 females): non-adverse

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (not-significant) in females: non-adverse
100 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (significant) in females: non-adverse
400 mg/kg bw/day: increased monocyte level in males (not significant); decrease in segmented neutrophils and increase in lymphocyte relative level (not significant) in females; increased granulocytes count and decreased lymphocyte level in females of the recovery group: non-adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
100 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
400 mg/kg bw/day: lower calcium concentration in males (not significant); decreased chloride concentration in females (significant); decreased inorganic phosphates concentration and decreased total creatinine kinase after 2 weeks of recovery in females; increased triglycerides in males and females (not significant); decrease in glutamate dehydrogenase (not significant): non-adverse

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day:
- increased liver weight in males and females compared to control animals, associated with microscopic findings
- slightly increased testis weight, non-significant, unclear relationship with test item administration
- decreased testicular and seminal vesicle weight in males of the recovery group, accompanied by an increase in the glans penis weight
- slight decrease in the relative weight of the spleen in males of the recovery group

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day:
- one large incapsulated kidney in 1/12 females (microscopic analysis revealed aseptic hydronephrosis), incidential, non-adverse;
- deformed spleen in 1/12 males, associated with a slightly increased spleen weight, lack of correlation to microscopic findings, unclear relationship with test item administration

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day:
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 2/6 females (also found in 1/6 control females), uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

100 mg/kg bw/day:
- Multifocal hepatocellular hypertrophy in 2/6 males
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 3/6 females (also found in 1/6 control females), one female had severe unilateral hydronephrosis, uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

400 mg/kg bw/day:
- Generalized hepatocellular hypertrophy (slight grade) in 4/6 males, statistically significant
- Hepatocellular hypertrophy, higher liver weight and glycogen accumulation in females, adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (statistically significant in males), test item related but species specific, non-adverse;
- Mineralization of kidney tubules in 1/6 males and 2/6 famles, uncertain relationship to test item
- Atrophy and fibrosis of red pulp in the spleen in 2/6 males, not correlating to hematological changes and changes in spleen weight
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, focal degeneration of epithelium in non-glandular stomach in 1/6 females, uncertain relationship to test item
- Significantly higher relative count in granulocytes (p < 0.05) in bone marrow smear in females (satellite group), due to the increase in the level of segmented neutrophils, findings correlated to the granulocyte absolute and relative count in peripheric blood after recovery period, non-adverse
- Slightly reduced erythrocariocytes in bone marrow smear of females (satellite group), leading to an increased myeloid-erythroid ratio, non-adverse

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

MORBIDITY AND MORTALITY
There was no morbidity and mortality of animals caused by the test item administration.

CLINICAL SIGNS:
Excessive salivation was observed 15 min after administration in 4/17 males and 13/17 females treated at 400 mg/kg bw/day, starting from the third week of dosing. Transient salivation following dosing is considered to be a sign of an adverse test item taste or a locally irritating effect. It is not considered toxicologically relevant.
In addition, 3 females of the high dose group had chromodacryrrhea starting from the 7th week of administration with an associated unilateral palpebral closure noted for one of them. Despite its low frequency, chromodacryorrhea is considered to be related to the test item administration.
Transient nasal red-brown discharge/crust was observed in one female treated at 25 mg/kg bw/day and one male and two females in the 400 mg/kg bw/day dose group. In one female nasal discharge was associated with the short-term rales. In addition, rales were noted for one female in the 25 mg/kg bw/day dose group. Incidents of nasal discharge and rales can be caused by aspiration of formulation, were not correlated with test item related microscopic findings and not considered to be toxicologically relevant. One male treated at 25 mg/kg bw/day had clinical signs of slight distress including piloerection, reactivity to handling and contraction of whisker pad starting , starting from the end of the second treatment week. No apparent cause of the poor clinical condition was determined. Clinical observations were not associated with any gross findings and significant microscopic changes in selected organs.
In the 400 mg/kg bw/day dose, the test item related piloerection and reactivity to handling were observed in individual females (No. 138 and No. 98, respectively) by the end of the administration period. Besides, piloerection was noted for a single male (No.6) in the 100 mg/kg bw/day dose group.

BODY WEIGHT AND WEIGHT CHANGES:
Mean body weights and body weight gains were unaffected by the test item for all study periods. .

FOOD CONSUMPTION:
Mean food consumption of animals of all treatment groups was similar to that in the vehicle control group for any treatment periods.

FUNCTIONAL OBSERVATION BATTERY:
Home cage, handling, open field, sensory, neuromuscular and physiological parameters evaluated during Functional Observation Battery testing as well as locomotor activity patterns were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the vehicle control group on study Day 27 (males), on PND 13 (females) or after a two-week recovery period.

HAEMATOLOGICAL FINDINGS:
The test item significantly increased the level of monocytes in males of dosage group 25 and 100 mg/kg bw/day and showed a similar trend (not significant) in animals treated at 400 mg/kg bw/day. The effect was considered treatment related, but non-adverse, as a dose-relationship was lacking and no changes in bone marrow were observed. After two weeks of recovery, hematological parameters in the 400 mg/kg bw/day male group were comparable with values in the control group. In blood smear of females, a statistically significant test-item related decrease in segmented neutrophils and an increase in relative lymphocyte relative level (p < 0.05) was observed in the 100 mg/kg bw/day dose group. The same trend was observed for females treated at 25 and 400 mg/kg bw/day, but without statistical significance.
Females of the 400 mg/kg bw/day dose recovery group exhibited, on the contrary, an increase in granulocytes count with a decrease in lymphocytes level, due to the recovery of the described above changes. Changes in the blood of the 400 mg/kg bw/day recovery females were correlated to the findings in the bone marrow smear. Thus, the hematological changes in females were slight, reversible and considered as non-adverse.

CLINICAL BIOCHEMISTRY:
A statistically significant decrease in calcium concentration was observed in males treated at 25 and 100 mg/kg bw/day (by 4.1%, p < 0.01) and a non-significant decrease in males of the 400 mg/kg bw/day dose group (by 1.7%). However, after the 2-week post-treatment period in this group, calcium concentration was increased compared with the control group, probably due to a recovery rebound effect. In addition, a slight (2.6%) but statistically significantly reduced chloride concentration (p < 0.05) was observed in females treated at 400 mg/kg bw/day. The effect did not correlate to the microscopic changes in kidneys and was thus considered as non-adverse.
Decreased inorganic phosphate and decreased total creatinine kinase activity was observed in females of the 400 mg/kg bw/day post-treatment subgroup and attributed to an impaired phosphate exchange.
In addition, increased triglycerides values were observed in males and females of the high dose group and may be associated with microscopic findings in the liver. The findings were slight, statistically non-significant and not considered adverse.
Slightly decreased glutamate dehydrogenase activity in animals treated at 400 mg/kg bw/day were not considered toxicologically relevant, as there was no statistical significance and other liver parameters were not found to be altered.

URINALYSIS:
A slightly higher mean diuresis value (non-significant) was observed in animals treated at 400 mg/kg bw/day. The effect was considered not test item-related.
There were no statistically significant differences for urinalysis parameters in F0 males at the end of the 28-day treatment period when the vehicle control and test item-treated groups were compared. After the 2-week post-treatment period, urinalysis values in 400 mg/kg bw/day dose group were also similar with values in a vehicle control group.
Analysis of creatinine in urine did not reveal significant changes in creatinine concentration in all dose treated males a the and of 28-day administration period as well as after 2-week post-treatment period.

ORGAN WEIGHT FINDINGS:
Increased absolute organ weights were found only in animals of the high dose group (400 mg/kg bw/day). An increased liver weight was observed in males (14.6%, p < 0.05) and females (12.1%, p < 0.05) when compared to control animals. The effect was associated with microscopic findings and considered test-item related. After the 400 mg/kg bw/day dose post treatment period, the absolute and relative liver weight in males and females did not significantly differ from the values in the control group.
In 400 mg/kg bw/day recovery subgroup males, a decrease in absolute and relative weights of testes was observed (p < 0.05). Furthermore, there was a tendency to a reduction in the relative weight of the seminal vesicles. The decrease in the testicular and seminal vesicles weight in recovery males was accompanied by an increase in the weight of the glans penis, which was statistically significant as compared with the control group for the absolute value (by 11.7%, p < 0.05). In contrast, during the administration period, there was a slight increase in the mean value of the testes weight noticeable for the right of the pair (by 12.3 % for absolute value, and by 11.7 % relative to body weight value). However, change in the weight of testes in the treatment period was not significant, was not associated with the microscopic findings for both treatment and post-treatment period, and therefore is regarded as change with an unclear relationship with the test item administration. In addition, the slight decrease in the relative weight of spleen was observed in the 400 mg/kg bw/day post-treatment males. This observation with an unclear relationship with the test item administration did not correlate to the microscopic findings in the spleen for post-treatment males. At the same time, in males of the 400 mg/kg bw/day main subgroup, incidents of atrophy and fibrosis were found in the spleen without any notable changes in the organ weight.

GROSS PATHOLOGICAL FINDINGS:
One female in the 100 mg/kg bw/day dose group had one large incapsulated kidney, that was associated with the microscopic findings (aseptic hydronephrosis). This finding was considered incidental and most likely not related to the test item administration.
One male in the 100 mg/kg bw/day dose group had a visually deformed spleen. This gross observation was associated with a slight increase in individual spleen weight, was not correlated to the microscopic findings in the spleen of males necropsied after 28 days of test item treatment. The spleen is regarded as an organ with an unclear relationship with the test item administration.
Macroscopic findings in thymus, lymph nodes, thyroids, adrenals, and pituitary concerned visual changes in organs size, occurred at similar frequencies in the test item and the vehicle control groups, did not correlate with a statistical change in the weights, and were associated with individual variability in organs size or weights.
Gross finding in urinary bladder (opaque, dense area) was noted in some males and females including the control group, was not correlated to the changes in organ weight or any microscopic observations, and thus considered not to be test item-related.

HISTOPATHOLOGICAL FINDINGS, NON-NEOPLASTIC:
Test item-related findings were observed in the liver, the thyroid glands and in the bone marrow. Findings with unclear relation to the administration of the test item were observed in kidneys, spleen and stomach.
Multifocal hepatocellular hypertrophy was noted in two of six examined males at 100 mg/kg bw/day and generalized hepatocellular hypertrophy of slight grade was observed in four of six males at 400 mg/kg bw/day. In the high dose group, the incidence of observation was statistically higher compared to the control vehicle group (p < 0.05).
In females, hepatocellular hypertrophy was found in all groups, including the vehicle control group, but was not observed in females of the control satellite subgroup and can be due to the physiological state of pregnancy and lactation. However, in all examined females at 25, 100 and 400 mg/kg bw/day hepatocellular hypertrophy was of more severe grade compared to the vehicle control group. Hepatocellular hypertrophy correlated to the increased absolute and relative liver weights and clinical pathology changes, and can be associated with microsomal enzyme induction. Besides, cytoplasmic alteration (glycogen accumulation) of slight to marked grade was found in a half of examined females of all dose treated group. Hepatocellular hypertrophy and higher liver weights as well as glycogen accumulation in the cytoplasm of hepatocytes were reversible, considered adaptive changes and hence non-adverse.
Epithelial hypertrophy in follicles of the thyroid gland was observed in some males and females in all test item-treated groups. In males at 400 mg/kg bw/day, an increase in finding frequency was statistically significant (p < 0.05). Follicular epithelial hypertrophy can occur with enzyme induction and increased hepatic clearance of thyroxin, was reversible and is considered to be species-specific and non-adverse. In parental animals, microscopic findings in thyroid gland were not correlated to changes in the thyroxin level as well as to the changes in organ weight. There were no test item-related findings in thyroid gland in 400 mg/kg bw/day post-treatment animals. At the same time, a lower level of thyroxin associated with increased thyroids weight was revealed in F1 offspring. So, the changes in the thyroid gland of parental rats are considered as test item-related; however, are considered to be species-specific and non-adverse.
Findings of uncertain relationship to the test item included mineralization of the tubules in the kidneys. Mineralization of tubules in papilla or inner stripe of the outer medulla was found in 1 of 6 females in the control group, 2 of 6 females in the 25 mg/kg bw/day group, 3 of 6 females in the 100 mg/kg bw/day group and 2 of 6 females in the 400 mg/kg bw/day group, and in single male in the 400 mg/kg bw/day group. Mineralization in the corticomedullary junction is a common finding in rats. There was no statistically significant increase in the frequency of mineralization. However, in one female in the 100 mg/kg bw/day group the severe unilateral hydronephrosis was revealed, which possibly could have been caused by mineralization and obstruction of the urinary tract. The signs of chronic progressive nephropathy were observed in approximately similar frequency in males and less often in females of all groups including the control group.
In two males from the 400 mg/kg bw/day group, atrophy, and fibrosis of red pulp in the spleen with or without atrophy of white pulp was observed. These findings did not correlate to the hematological changes and changes in spleen weight. The slight decrease in the mean relative weight of spleen was noted for males in the 400 mg/kg bw/day post-treatment group; however, this change was not statistically significant compared to the control group.
In the forestomach of single males from of each dose group, the neutrophilic or macrophage infiltration of deep mucosa and/or submucosa of the minimum and moderate grade was found with concomitant focal degeneration of epithelium in one male. Besides, the focal degeneration of epithelium in nonglandular stomach was observed in one female in the 400 mg/kg bw/day group. Observations in the forestomach can potentially indicate a local effect of the test item administered by gavage. However, these findings were incidental and slight, and the relationship with the test item is considered unclear.
No test item-related histological findings in organs of the male and female reproductive system were revealed. The decrease in absolute and relative weights of testes and a tendency to a reduction in the relative weight of the seminal vesicles in the 400 mg/kg bw/day recovery males did not correlate to any microscopic changes in these organs in the test item treated males euthanized on day 29 or after 2-weeks post-treatment period.
During qualitative examination of the testes with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure, no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed. In the 400 mg/kg bw/day dose group, inflammation in the tunica albuginea of one testis was observed in single male. In another male, chronic inflammation in the ventral lobe of the prostate was found. These findings were of minimal and slight grade and considered to be not test item-related.
In ovaries and oviducts of the females, there were no significant microscopic changes that could be associated with the test item. Findings in the uterus and vagina, observed with a similar frequency in the control group and 400 mg/kg bw/day group, are assumed to be related to parturition.
There were no significant abnormalities in the ovaries, oviducts, and uterus of non-gravid females No.103 (group 3) and No.138 (group 4), which could have affected the ability of these females for pregnancy. However, in the female No.138, findings in liver were observed, which are supposed to be associated with the test item administration (glucogen accumulation of minimal grade and pigment in Kupffer cells of moderate grade). Microscopic alterations in bone marrow smear were observed only in 400 mg/kg bw/day dose post-treatment females and comprised a significantly higherrelative count in granulocytes (p < 0.05) due to the increase in the level of segmented neutrophils. The findings correlated to the granulocyte absolute and relative count in peripheric blood after the recovery period and were considered as non-adverse. In addition, in this female dose group the percentage of erythrocariocytes was slightly reduced leading to an increased myeloid-erythroid ratio (non-adverse).

Dose descriptor:

NOAEL

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at highest dose

Critical effects observed:

no

Conclusions:

Based on the results of this study, the no observed adverse effect level (NOAEL) for systemic toxicity was ≥ 400 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service, Bratislava, Slovak Republic

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trihexyl phosphate

EC Number:

219-774-8

EC Name:

Trihexyl phosphate

Cas Number:

2528-39-4

Molecular formula:

C18H39O4P

IUPAC Name:

trihexyl phosphate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

outbreed

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Pushchino, Russia
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 348 ± 27 g (males) and 213 ± 14 g (females)
- Housing: Animals were housed in groups of 2/sex in polycarbonate cages type IV (595 x 380 x 200 mm) until mating. During the mating period, males were housed alone and cohabited with females in the home cage of the male. After mating, females were housed individually in these cages. Males and females of the satellite group (not mated) were housed 2-3 animals per cage. Commercial autoclaved woodchip bedding was used.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300, Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water: filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 30 - 63
- Air changes (per hr): at least 12 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Aug 2018 To: 09 Jan 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Required volumes of the test item (taking into account the purity and the density of the liquid) were mixed with the calculated volume of vehicle (water) and stirred to create a homogeneous water emulsion. Dosing formulations were prepared every three days, aliquoted to the required volumes of days of administration and stored at room temperature in tightly closed glass jars in the fume hood. On the day of dosing, the aliquot of each formulation was mixed thoroughly and stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 5, 20 and 80 mg/mL
- Amount of vehicle: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: presence of sperm following vaginal lavage termed gestation day 0
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations were analysed for homogeneity, stability and concentration. Stability of the test item in the vehicle prepared at concentrations of 5, 20 and 80 mg/mL was confirmed following 3 days of storage at room temperature during method validation study. Additional data on the stability of dose formulations were obtained by analyzing the medium strata of all dose formulations after their 2-day storage and resuspension at the beginning of in-life phase of the study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a LC/MS validated method at the beginning, in the middle and at the end of in-life phase. In addition, homogeneity analysis was performed at the beginning of the study after 2 days of dose storage after resuspension.
For stability, the mean concentration of formulation samples of 5 and 80 mg/mL after 3 days of storage and resuspension should be within the acceptable range of the target concentration (85 – 115% with RSD < 10%), and within the range 85-115% of the time zero point.
Stability of the test substance formulations was confirmed after three days of storage at room temperature and after 2-day storage incl. resuspension, except for dose group 2 (20 mg/mL) which exceeded the acceptable range (120.5%) presumably due to problems of homogenization and sample preparing.
For dose formulations 5, 20 and 80 mg/mL all analysed concentrations were within the acceptable range of 85 – 115% of the target concentration for all dates of analysis, excluding formulation 20 mg/mL on date of 27 Nov 2018, for which the actual concentration was 81.0% of the target level.
The samples were considered homogeneous since the relative standard deviation (RSD) for the mean concentration was ≤ 10% at a concentration within the acceptable limits (85 – 115% of target concentration) with the following exceptions: The RSD value exceeded 10% on date of 27 Nov 2018 for 20 mg/mL (10.2%) and 80 mg/mL (12%) with a mean concentration for 20 mg/mL formulation less than the acceptable limit (79.6%). The homogeneity data of the formulations after 2-day storage and re-mixing were outside the acceptable range for the formulation of 5 mg/mL (RSD 10.9%) and 20 mg/mL (RSD 10.2% and mean concentration 117.8% of target). However, the described outputs from the acceptable range were slight, and the deviations are considered to be mild. In general, it can be concluded that dose formulations homogeneity was acceptable, not affecting the interpretation of calculated dosages administered to animals.

Duration of treatment / exposure:

Males: Treatment for 28 days, beginning 14 days prior to mating, throughout the mating period until necropsy
Females: Females which delivered were treated for 49 – 56 days, beginning 14 days prior to mating until lactation day 13. Females which failed to deliver/ with no evidence of mating were treated for 55 days.
Animals of the recovery group were not mated and dosed for 28 days (males) or 55 days (females) with following two weeks recovery period.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

not applicable for an OECD 422 study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12 (main group)
5 (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Available toxicity data of structurally similar analogue substances revealed that doses of the test item used in the study on repeated dose toxicity can be in the range of 25 to 400 mg/kg bw/day. A pilot dose-range finding study with the test item was performed and confirmed that a dose of 400 mg/kg bw/day will be tolerated without causing death or obvious suffering. Test item doses of 25 and 400 mg/kg bw/day were administered daily for 21 days to each three male and three female Sprague Dawley rats. No mortalities and no adverse clinical signs were observed, except for excessive salivation in one female treated at 400 mg/kg bw/day. No effects on food consumption and body weight gain was noted and there were no gross macroscopic findings at necropsy. Increased liver weights were observed in males at 25 and 400 mg/kg bw/day and in females at 400 mg/kg bw/day and assumed to be caused by hepatocellular hypertrophy. Based on these results and based on the published data of analogues toxicity, doses of 25, 100 and 400 mg/kg bw/day were chosen for the current study..

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/morbidity
- Cage side observations included: All animals were observed for general condition and clinical signs of toxicity 5 - 20 min following dose administration. Females expected to deliver were observed twice daily for dystocia and other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and on a weekly basis throughout the study
- Observations included: chromodacryorrhea, palpebral aperture, hunching, piloerection, respiration, alertness, reactivity to handling, vocalization, mucous membranes, locomotion/hypokinesia, gait and stereotypes/bizzares

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on Day 1 prior to dosing and weekly thereafter. During pregnancy, mated females were weighed on Days 0, 7, 14, 20 and within 24 h of parturition and on post-natal days (PND) 2, 4 and 13.

FOOD CONSUMPTION: Yes
- Food consumption was measured prior to dosing and weekly until cohabition. Food consumption was not recorded during mating. After mating, food consumption of females was measured on gestation Days 0-1, 6-7, 13-14 and 19-20 and on lactation Days 1-2, 4-5 and 12-13.

WATER CONSUMPTION: No

OTHER:
Haematology and coagulation, clinical chemistry, urinalysis, neurobehavioral examination and thyroid hormone analysis (for details refer to IUCLID section 7.5.1)

Oestrous cyclicity (parental animals):

Estrous cyclicity was monitored by daily vaginal lavage for 14 days during the pre-mating period and during the mating period until evidence of mating was observed. On the day of necropsy, vaginal smears were examined to determine the stage of the estrous cycle and allow correlation with histopathology of female reproductive organs.

Sperm parameters (parental animals):

Parameters examined male parental generations (P0):
testis weight, epididymis weight, seminal vesicles and coagulation glands weight, prostate gland weight, histopathological examination of epididymides, prostate, seminal vesicles and coagulating glands
A detailed qualitative examination of the testes was done with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. The examination was conducted in order to indentify treatment-related effects such as missing germ cell layers, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatigenic cells into the lumen.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups on post-natal day (PND) 13, organ weight of thyroid glands (1/sex)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; pups not surviving to the scheduled necropsy were examined for floating of the lungs and the presence of milk in the stomach. If possible, defects or cause of death were evaluated.

OTHER:
THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood samples were collected on PND 4 and at termination on PND 13
- Anaesthetic used for blood collection: Yes (Zoletil® and Xyla® (tiletamine+zolazepam+xylazine))
- Animals fasted: No
- How many animals: 2 pups per litter
- Parameters examined: thyroxine (T4)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were euthanized by anesthesia (Zoletil® / Xyla®) following completion of the mating period and 28 days of dose administration.
- Female animals: All surviving dams were euthanized on lactation Day 14 after litters have been euthanized on post-natal Day (PND) 13. Females which have not delivered were euthanized after 55 days of dosing. All females were euthanized by anesthesia (Zoletil® / Xyla®).
- Satellite animals: All satellite animals were subjected to euthanasia at the end of two weeks post-treatment period. All animals were euthanized by anesthesia (Zoletil® / Xyla®).

GROSS NECROPSY
- Gross necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: adrenals, brain, cowper’s glands, epididymides, heart, glans penis, kidneys, levator ani and bulbocavernosus muscles complex, liver, ovaries, pituitary, prostate, seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid and uterus. The tissues indicated in Table No. 1 were prepared for light microscopic examination from six randomly selected adult males and females in the control and high-dose groups. Liver, kidneys, spleen, stomach, testes, epididymides, and seminal vesicles with coagulating glands were examined in the low- and mid dose groups as well as in satellite subgroup animals. Microscopic examination of thyroid glands was done for all adult males and femlaes in all groups including satellite subgroups, and one male and female of day 13 pup from each litter in all groups.

Postmortem examinations (offspring):

SACRIFICE
At scheduled euthanasia on post-natal Day (PND) 13 two pups (1/sex) from each litter were anesthetized (Zoletil® / Xyla®) and euthanized by terminal blood collection. All other pups from litter were euthanized on PND 13 by carbon dioxide inhalation.
The animals were subjected to postmortem examination as follows:

GROSS NECROPSY
All surviving pups were subjected to gross necropsy with examination for gross abnormalities and particular attention to the external reproductive genitals.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands from pups were weighed and microscopically examined (one male and one female from each litter).

Statistics:

All statistical tests were performed separately for each sex and compared the treated groups to the control group.
- Multi-factor analysis of variance ANOVA-2, followed by the Duncan test: mean body weights, body weight gain and food consumption data
- Parametric one-way analysis of variance (ANOVA), followed by Dunnett's test in case of significance (p < 0.05): estrus cycle length, pre-coital intervals, gestation length, former implantation sites, clinical pathology values and functional observational battery (FOB) data, organ weights (absolute and relative to body weights and relative to brain weights), mean litter weights, live litter size, and a number of pups born
- T-test: clinical pathology values and FOB values of satellite animals, comparison of organ weights
- Fisher’s exact test: FOB parameters which yield scalar or descriptive data, histopathological findings
- Kruskal-Wallis nonparametric ANOVA test: pup viability and sex ratio data
- Kruskal-Wallis nonparametric ANOVA test with Dunn’s test: gamma-glutamyltransferase data
- Chi-square test: male copulation, female conception, male and female mating and fertility indices of the treated groups

Reproductive indices:

Male mating index (%) = (Number of males with evidence of mating/Total number of males used for mating) x 100
Female mating index (%) = (Number of females with evidence of mating/Total number of females used for mating) x 100
Male fertility index (%) = (Number of males siring a litter/Total number of males used for mating) x 100
Male corpulation index (%) = (Number of males siring a litter/Number of males with evidence of mating or females with confirmed pregnancy) x 100
Female fertility index (%) = (Number of females with confirmed pregnancy/Total number of females used for mating) x 100
Female conception index (%) = (Number of females with confirmed pregnancy/Number of females with evidence of mating) x 100
Gestation index (%) = Number of rats with live offspring/Number of pregnant rats

Offspring viability indices:

Postnatal survival (% per litter)= Number of live pups on PND 0-1, PND 1-4, PND 1-7, PND 1-13
Number of dead pups (%) = Pups found dead, euthanized in extremis and cannibalized on PND 0-1, PND 1-4, PND 1-7, PND 1-13
Viability index (%) = Number of live pups/Number of liveborn pups on PND 0, 4, 7 and 13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: piloerection, reactivity to handling and contraction of whisker pad (1/12 males); nasal red-brown discharge/crust and rales (1/12 females): non-adverse
100 mg/kg bw/day: nasal red-brown discharge/crust (1/12 males), transient effect; piloerection (1/12 males): non-adverse
400 mg/kg bw/day: excessive salivation 15 min after administration (4/17 males, 13/17 females); chromodacryorrhea (3/17 females); piloerection and reactivity to handling (2/17 females); nasal red-brown discharge/crust (1/17 males, 2/17 females): non-adverse

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (not-significant) in females: non-adverse
100 mg/kg bw/day: significant monocytosis in males; decrease in segmented neutrophils and increase in lymphocyte relative level (significant) in females: non-adverse
400 mg/kg bw/day: increased monocyte level in males (not significant); decrease in segmented neutrophils and increase in lymphocyte relative level (not significant) in females; increased granulocytes count and decreased lymphocyte level in females of the recovery group: non-adverse

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
100 mg/kg bw/day: lower calcium concentration in males (significant): non-adverse
400 mg/kg bw/day: lower calcium concentration in males (not significant); decreased chloride concentration in females (significant); decreased inorganic phosphates concentration and decreased total creatinine kinase after 2 weeks of recovery in females; increased triglycerides in males and females (not significant); decrease in glutamate dehydrogenase (not significant): non-adverse

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day:
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 2/6 females (also found in 1/6 control females), uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

100 mg/kg bw/day:
- Multifocal hepatocellular hypertrophy in 2/6 males
- Hepatocellular hypertrophy, glycogen accumulation in females: adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (not significant), test item related but species specific, non-adverse
- Mineralization of kidney tubules in 3/6 females (also found in 1/6 control females), one female had severe unilateral hydronephrosis, uncertain relationship to test item
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, slight and incidental, uncertain relationship to test item

400 mg/kg bw/day:
- Generalized hepatocellular hypertrophy (slight grade) in 4/6 males, statistically significant
- Hepatocellular hypertrophy, higher liver weight and glycogen accumulation in females, adaptive changes, non-adverse
- Epithelial hypertrophy in follicles of the thyroid gland (statistically significant in males), test item related but species specific, non-adverse;
- Mineralization of kidney tubules in 1/6 males and 2/6 famles, uncertain relationship to test item
- Atrophy and fibrosis of red pulp in the spleen in 2/6 males, not correlating to hematological changes and changes in spleen weight
- Neutrophilic or macrophage infiltration of deep mucosa and/or submucosa in the forestomach of single males, focal degeneration of epithelium in non-glandular stomach in 1/6 females, uncertain relationship to test item
- Significantly higher relative count in granulocytes (p < 0.05) in bone marrow smear in females (satellite group), due to the increase in the level of segmented neutrophils, findings correlated to the granulocyte absolute and relative count in peripheric blood after recovery period, non-adverse
- Slightly reduced erythrocariocytes in bone marrow smear of females (satellite group), leading to an increased myeloid-erythroid ratio, non-adverse

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: irregular cycle in 1/12 females, did not affect the onset of pregnancy

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day:inflammation in the tunica albuginea of one testis in 1/12 males; chronic inflammation in the ventral lobe of the prostate in another male

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

100 and 400 mg/kg bw/day: each one male did not sire a litter; each one female were not pregnant without implantation sites.
100 mg/kg bw/day: one female had prolonged labor with bleeding and cannabilization of 5 from 11 delivered pups, associated with incidental kidney damage in this female

Details on results (P0)

MORBIDITY AND MORTALITY
There was no morbidity and mortality of animals caused by the test item administration.

CLINICAL SIGNS:
Excessive salivation was observed 15 min after administration in 4/17 males and 13/17 females treated at 400 mg/kg bw/day, starting from the third week of dosing. Transient salivation following dosing is considered to be a sign of an adverse test item taste or a locally irritating effect. It is not considered toxicologically relevant.
In addition, 3 females of the high dose group had chromodacryrrhea starting from the 7th week of administration with an associated unilateral palpebral closure noted for one of them. Despite its low frequency, chromodacryorrhea is considered to be related to the test item administration.
Transient nasal red-brown discharge/crust was observed in one female treated at 25 mg/kg bw/day and one male and two females in the 400 mg/kg bw/day dose group. In one female nasal discharge was associated with the short-term rales. In addition, rales were noted for one female in the 25 mg/kg bw/day dose group. Incidents of nasal discharge and rales can be caused by aspiration of formulation, were not correlated with test item related microscopic findings and not considered to be toxicologically relevant. One male treated at 25 mg/kg bw/day had clinical signs of slight distress including piloerection, reactivity to handling and contraction of whisker pad starting , starting from the end of the second treatment week. No apparent cause of the poor clinical condition was determined. Clinical observations were not associated with any gross findings and significant microscopic changes in selected organs.
In the 400 mg/kg bw/day dose, the test item related piloerection and reactivity to handling were observed in individual females (No. 138 and No. 98, respectively) by the end of the administration period. Besides, piloerection was noted for a single male (No.6) in the 100 mg/kg bw/day dose group.
BODY WEIGHT AND WEIGHT CHANGES:
Mean body weights and body weight gains were unaffected by the test item for all study periods. .

FOOD CONSUMPTION:
Mean food consumption of animals of all treatment groups was similar to that in the vehicle control group for any treatment periods.

FUNCTIONAL OBSERVATION BATTERY:
Home cage, handling, open field, sensory, neuromuscular and physiological parameters evaluated during Functional Observation Battery testing as well as locomotor activity patterns were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the vehicle control group on study Day 27 (males), on PND 13 (females) or after a two-week recovery period.

HAEMATOLOGICAL FINDINGS:
The test item significantly increased the level of monocytes in males of dosage group 25 and 100 mg/kg bw/day and showed a similar trend (not significant) in animals treated at 400 mg/kg bw/day. The effect was considered treatment related, but non-adverse, as a dose-relationship was lacking and no changes in bone marrow were observed. After two weeks of recovery, hematological parameters in the 400 mg/kg bw/day male group were comparable with values in the control group. In blood smear of females, a statistically significant test-item related decrease in segmented neutrophils and an increase in relative lymphocyte relative level (p < 0.05) was observed in the 100 mg/kg bw/day dose group. The same trend was observed for females treated at 25 and 400 mg/kg bw/day, but without statistical significance.
Females of the 400 mg/kg bw/day dose recovery group exhibited, on the contrary, an increase in granulocytes count with a decrease in lymphocytes level, due to the recovery of the described above changes. Changes in the blood of the 400 mg/kg bw/day recovery females were correlated to the findings in the bone marrow smear. Thus, the hematological changes in females were slight, reversible and considered as non-adverse.

CLINICAL BIOCHEMISTRY:
A statistically significant decrease in calcium concentration was observed in males treated at 25 and 100 mg/kg bw/day (by 4.1%, p < 0.01) and a non-significant decrease in males of the 400 mg/kg bw/day dose group (by 1.7%). However, after the 2-week post-treatment period in this group, calcium concentration was increased compared with the control group, probably due to a recovery rebound effect. In addition, a slight (2.6%) but statistically significantly reduced chloride concentration (p < 0.05) was observed in females treated at 400 mg/kg bw/day. The effect did not correlate to the microscopic changes in kidneys and was thus considered as non-adverse.
Decreased inorganic phosphate and decreased total creatinine kinase activity was observed in females of the 400 mg/kg bw/day post-treatment subgroup and attributed to an impaired phosphate exchange.
In addition, increased triglycerides values were observed in males and females of the high dose group and may be associated with microscopic findings in the liver. The findings were slight, statistically non-significant and not considered adverse.
Slightly decreased glutamate dehydrogenase activity in animals treated at 400 mg/kg bw/day were not considered toxicologically relevant, as there was no statistical significance and other liver parameters were not found to be altered.

URINALYSIS:
A slightly higher mean diuresis value (non-significant) was observed in animals treated at 400 mg/kg bw/day. The effect was considered not test item-related.
There were no statistically significant differences for urinalysis parameters in F0 males at the end of the 28-day treatment period when the vehicle control and test item-treated groups were compared. After the 2-week post-treatment period, urinalysis values in 400 mg/kg bw/day dose group were also similar with values in a vehicle control group.
Analysis of creatinine in urine did not reveal significant changes in creatinine concentration in all dose treated males a the and of 28-day administration period as well as after 2-week post-treatment period.

ORGAN WEIGHT FINDINGS:
Increased absolute organ weights were found only in animals of the high dose group (400 mg/kg bw/day). An increased liver weight was observed in males (14.6%, p < 0.05) and females (12.1%, p < 0.05) when compared to control animals. The effect was associated with microscopic findings and considered test-item related. After the 400 mg/kg bw/day dose post treatment period, the absolute and relative liver weight in males and females did not significantly differ from the values in the control group.
In 400 mg/kg bw/day recovery subgroup males, a decrease in absolute and relative weights of testes was observed (p < 0.05). Furthermore, there was a tendency to a reduction in the relative weight of the seminal vesicles. The decrease in the testicular and seminal vesicles weight in recovery males was accompanied by an increase in the weight of the glans penis, which was statistically significant as compared with the control group for the absolute value (by 11.7%, p < 0.05). In contrast, during the administration period, there was a slight increase in the mean value of the testes weight noticeable for the right of the pair (by 12.3 % for absolute value, and by 11.7 % relative to body weight value). However, change in the weight of testes in the treatment period was not significant, was not associated with the microscopic findings for both treatment and post-treatment period, and therefore is regarded as change with an unclear relationship with the test item administration. In addition, the slight decrease in the relative weight of spleen was observed in the 400 mg/kg bw/day post-treatment males. This observation with an unclear relationship with the test item administration did not correlate to the microscopic findings in the spleen for post-treatment males. At the same time, in males of the 400 mg/kg bw/day main subgroup, incidents of atrophy and fibrosis were found in the spleen without any notable changes in the organ weight.

GROSS PATHOLOGICAL FINDINGS:
One female in the 100 mg/kg bw/day dose group had one large incapsulated kidney, that was associated with the microscopic findings (aseptic hydronephrosis). This finding was considered incidental and most likely not related to the test item administration.
One male in the 100 mg/kg bw/day dose group had a visually deformed spleen. This gross observation was associated with a slight increase in individual spleen weight, was not correlated to the microscopic findings in the spleen of males necropsied after 28 days of test item treatment. The spleen is regarded as an organ with an unclear relationship with the test item administration.
Macroscopic findings in thymus, lymph nodes, thyroids, adrenals, and pituitary concerned visual changes in organs size, occurred at similar frequencies in the test item and the vehicle control groups, did not correlate with a statistical change in the weights, and were associated with individual variability in organs size or weights.
Gross finding in urinary bladder (opaque, dense area) was noted in some males and females including the control group, was not correlated to the changes in organ weight or any microscopic observations, and thus considered not to be test item-related.

HISTOPATHOLOGICAL FINDINGS, NON-NEOPLASTIC:
Test item-related findings were observed in the liver, the thyroid glands and in the bone marrow. The findings with unclear relation to the administration of the test item were observed in kidneys, spleen and stomach.
Multifocal hepatocellular hypertrophy was noted in two of six examined males at 100 mg/kg bw/day and generalized hepatocellular hypertrophy of slight grade was observed in four of six males at 400 mg/kg bw/day. In the high dose group, the incidence of observation was statistically higher compared to the control vehicle group (p < 0.05).
In females, hepatocellular hypertrophy was found in all groups, including the vehicle control group, but was not observed in females of the control satellite subgroup and can be due to the physiological state of pregnancy and lactation. However, in all examined females at 25, 100 and 400 mg/kg bw/day hepatocellular hypertrophy was of more severe grade compared to the vehicle control group. Hepatocellular hypertrophy correlated to the increased absolute and relative liver weights and clinical pathology changes, and can be associated with microsomal enzyme induction. Besides, cytoplasmic alteration (glycogen accumulation) of slight to marked grade was found in a half of examined females of all dose treated group. Hepatocellular hypertrophy and higher liver weights as well as glycogen accumulation in the cytoplasm of hepatocytes were reversible, considered adaptive changes and hence non-adverse.
Epithelial hypertrophy in follicles of the thyroid gland was observed in some males and females in all test item-treated groups. In males at 400 mg/kg bw/day, an increase in finding frequency was statistically significant (p < 0.05). Follicular epithelial hypertrophy can occur with enzyme induction and increased hepatic clearance of thyroxine , was reversible and is considered to be species-specific and non-adverse (Curran P. and DeGroot L. Endocr Rev. 1991. 12(2):135-150). In parental animals, microscopic findings in thyroid gland were not correlated to changes in the thyroxin level as well as to the changes in organ weight. There were no test item-related findings in thyroid gland in 400 mg/kg bw/day post-treatment animals. At the same time, a lower level of thyroxin associated with increased thyroids weight was revealed in F1 offspring. So, the changes in the thyroid gland of parental rats are considered as test item-related; however, are considered to be species-specific and non-adverse.
Findings of uncertain relationship to the test item included mineralization of the tubules in the kidneys. Mineralization of tubules in papilla or inner stripe of the outer medulla was found in 1 of 6 females in the control group, 2 of 6 females in the 25 mg/kg bw/day group, 3 of 6 females in the 100 mg/kg bw/day group and 2 of 6 females in the 400 mg/kg bw/day group, and in single male in the 400 mg/kg bw/day group. Mineralization in the corticomedullary junction is a common finding in rats. There was no statistically significant increase in the frequency of mineralization. However, in one female in the 100 mg/kg bw/day group the severe unilateral hydronephrosis was revealed, which possibly could have been caused by mineralization and obstruction of the urinary tract. The signs of chronic progressive nephropathy were observed in approximately similar frequency in males and less often in females of all groups including the control group.
In two males from the 400 mg/kg bw/day group, atrophy, and fibrosis of red pulp in the spleen with or without atrophy of white pulp was observed. These findings did not correlate to the hematological changes and changes in spleen weight. The slight decrease in the mean relative weight of spleen was noted for males in the 400 mg/kg bw/day post-treatment group; however, this change was not statistically significant compared to the control group.
In the forestomach of single males from of each dose group, the neutrophilic or macrophage infiltration of deep mucosa and/or submucosa of the minimum and moderate grade was found with concomitant focal degeneration of epithelium in one male. Besides, the focal degeneration of epithelium in nonglandular stomach was observed in one female in the 400 mg/kg bw/day group. Observations in the forestomach can potentially indicate a local effect of the test item administered by gavage. However, these findings were incidental and slight, and the relationship with the test item is considered unclear.
No test item-related histological findings in organs of the male and female reproductive system were revealed. The decrease in absolute and relative weights of testes and a tendency to a reduction in the relative weight of the seminal vesicles in the 400 mg/kg bw/day recovery males did not correlate to any microscopic changes in these organs in the test item treated males euthanized on day 29 or after 2-weeks post-treatment period.
In ovaries and oviducts of the females, there were no significant microscopic changes that could be associated with the test item. Findings in the uterus and vagina, observed with a similar frequency in the control group and 400 mg/kg bw/day group, are assumed to be related to parturition.
There were no significant abnormalities in the ovaries, oviducts, and uterus of non-gravid females No.103 (group 3) and No.138 (group 4), which could have affected the ability of these females for pregnancy. However, in the female No.138, findings in liver were observed, which are supposed to be associated with the test item administration (glucogen accumulation of minimal grade and pigment in Kupffer cells of moderate grade). Microscopic alterations in bone marrow smear were observed only in 400 mg/kg bw/day dose post-treatment females and comprised a significantly higherrelative count in granulocytes (p < 0.05) due to the increase in the level of segmented neutrophils. The findings correlated to the granulocyte absolute and relative count in peripheric blood after the recovery period and were considered as non-adverse. In addition, in this female dose group the percentage of erythrocariocytes was slightly reduced leading to an increased myeloid-erythroid ratio (non-adverse).

REPRODUCTION FUNCTION: ESTROUS CYCLE
The duration of the estrous cycle, being evaluated during the administration period before the onset of pregnancy, was approximately the same in all groups. An irregular cycle was observed in one female treated at 100 mg/kg bw/day, which nevertheless did not affect the onset of pregnancy. Two females of the control group were cohabited with males during estrus and no estrus cycle was observed thereafter due to possible coitally induced pseudopregnancy. As a result, the animals were recorded as not-mated, non-gravid.

REPRODUCTION FUNCTION: SPERM MEASURES
During qualitative examination of the testes with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure, no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed. In the 400 mg/kg bw/day dose group, inflammation in the tunica albuginea of one testis was observed in single male. In another male, chronic inflammation in the ventral lobe of the prostate was found. These findings were of minimal and slight grade and considered to be not test item-related.

REPRODUCTION PERFORMANCE:
All twelve males in the 25, 100 and 400 mg/kg bw/day dose group were with evidence of mating and with pre-coital intervals that did not differ from the value in the control group. One male in the 100 mg/kg bw/day dose group and one male in the 400 mg/kg bw/day dose group did not sire a litter. Indexes of fertility and copulation were 91.7% in these groups, that was not different from the values in the control group. In the low dose group, the indices of fertility and copulation were 100.0%.
All females in all test item groups were with evidence of mating. In the 25 mg/kg bw/day dose group, 100.0% of females were gravid and delivered. One female in 100 mg/kg bw/day dose group and one female in the 400 mg/kg bw/day dose group were not pregnant without implantation sites. However, fertility and conception indices (91.7% in both groups) were comparable to the control group with a fertility index of 83.0% and conception index of 100.0%, respectively.
Mean gestation lengths were similar between all groups and most females had a gestation period of 22 days. Gestation indexes were 100.0% in all test item groups. There were no significant pre-delivery findings in any group. In the 100 mg/kg bw/day dose group, one female (No. 82) had prolonged labor with bleeding and cannibalization of 5 from 11 delivered pups. The disturbance in parturition can be associated with the kidney damage (hydronephrosis) have been founded during scheduled necropsy of this female. In other groups, abnormalities in the parturition were not observed.
The test item did not influence the pre-natal and post-natal loss of offspring by dams at any dosage level.
In the control group, two females had a small number of implantation sites and did not deliver a litter, which resulted in a slight decrease in the mean values of the number of implantation sites and liveborn pups compared to test item treated groups. However, the number of implantation sites and liveborn pups were not significantly different in the test item treated groups from the control group and were similar to the historical control data (MEAN±S.D., 12.6 ± 3.1 (N=35), implantation sites, and 11.3 ± 3.0 (N=34), liveborn pups).
The number of females with pre-natal loss of offspring was approximately the same between groups. Single females in each test item treated group had pups which died in the postnatal period. However, the post-natal death of offspring was low and was not test item-related. There were no dams with total litter loss and abnormal pups.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

25 mg/kg bw/day: 1 live born pup was found dead on post-natal day (PND) 1 with cyanosis of the pelvic area; one pup had dark spots on the head, neck and scapular area, incidental

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: increased number of cannibalized pups (3 litters affected)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: slight decrease in mean percentage values for body weight gain (not significant)
400 mg/kg bw/day: reduced absolute body weight of male and female pups (significant for females, not significant for males); significant decrease in the total absolute and percentage weight gain in both males and females

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:

effects observed, treatment-related

Description (incidence and severity):

25 mg/kg bw/day: 1-2 remaining areola in 1 male
400 mg/kg bw/day: increased frequency of areola retention (5 males with 1-2 remaining areola)
Females, all dose groups: extra areola/nipple count, attributed to genetic background of population

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: significantly increased relative weight of the thyroid glands in males and females

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

effects observed, non-treatment-related

Other effects:

effects observed, treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSIS:
100 mg/kg bw/day: significantly reduced serum concentration of thyroxin on PND 13
400 mg/kg bw/day: significantly reduced serum concentration of thyroxin on PND 4

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

CLINICAL SIGNS:
There were no clinical signs in pups in the post-natal period considered to be treatment-related. In the 25 mg/kg bw/day dose group one live born pup was found dead on post-natal day (PND) 1 with cyanosis of the pelvic area and one further pup had dark spots on the head, neck and scapular area starting PND 4 through PND 7. These findings were incidental, observed in the low dose group and not considered treatment-related.

MORTALITY:
There were no test item-related changes in the mean number of delivered, liveborn and stillborn pups. The mean absolute and percentage number of cannibalized pups was slightly increased in the 100 mg/kg bw/day dose group. Three litters in this group were with cannibalized newborns including litter No.82 with five partially cannibalized pups. Two of them were identified as stillborns, and other were with unknown vital status, but, it is also likely that they were stillborn. High mortality in the litter No.82 is presumably associated with the poor state of the female (severe unilateral hydronephrosis) and prolonged labor.
The mean values of litters survival on postnatal day 0 in all dose treated groups did not differ from the values in the control group.
The test item in all administrated doses did not affect the survival of the F1 offspring in the postnatal period. The mean number of liveborn pups, live litter size and postnatal survival in the all dose-treated groups did not significantly differ from the values in the control group. The percentage of dead offspring for the postnatal periods 1-4, 1-7, and 1-13 days, as well as viability indexes, were similar to the control group values.
Furthermore there were no test item-related mortalities during the post-natal period. One live born pup was found dead on post-natal day (PND) 1, which was associated with cyanosis of the pelvic area. The findings were incidental and not considered treatment-related.

BODY WEIGHT AND WEIGHT CHANGES:
Body weights at birth in test item treated groups did not differ significantly from the values in the control group.
In the 400 mg/kg bw/day dose group the absolute body weight of males and females were lower compared to control animals at post-natal day (PND) 13 (by 7.7% for males, not significant, and by 8.9% for females, p < 0.05). By PND 13, a significant decrease in the absolute and percentage weight gain was observed at 400 mg/kg bw/day in both males and females pups. A slight, non-significant decrease in the mean percentage values of body weight gain was also noted in the 100 mg/kg bw/day dose group. The percentage of males in all dosage groups did not significantly differ from the value in the control vehicle group.

SEXUAL MATURATION:
The absolute and normalized anogential distance in test item-treated groups did not differ significantly from the values in the control group.
On postnatal day 13, one male pup (1.8%) in the 25 mg/kg bw/day dose group and five male pups from three litters of the 400 mg/kg bw/day dose group had 1-2 remaining areolae (dark focal area that did not necessarily have a nipple bud that could be felt through the skin). The frequency of the areolae retention in the high dose group was 8.8% with the mean value of the number of areoles per male being slightly higher compared to the control group and historical control data. However, this change was not statistically significant. Nevertheless, the relationship of slight increase in nipples/areolae retention in PND 13 male pups with the test item at 400 mg/kg bw/day cannot be completely excluded.

ORGAN WEIGHT FINDINGS:
No statistically significant changes were found in the absolute weight of the thyroid glands in male and female offspring in all dose administrated groups. However, in the 400 mg/kg bw/day dose male group, the mean value of thyroids was slightly non-significantly increased (13.2 % compared to the control group). An increase in the relative weight of thyroid glands in this high dose group was significant in both male and female offspring compared with the control group (p < 0.05). The effect can be partially explained by the decrease in body weight of the pups in this group by postnatal day 13. However, a slight, not statistical significant tendency to an increase in the relative thyroids weight was already observed in the low dose group without apparent changes in the offspring body weight. The increase in thyroids weight was associated with a decrease in thyroxin level in PND 13 and PND 4 offspring at 100 and 400 mg/kg bw/day, respectively. No test item-related histological changes were observed in thyroid glands of male and female pups examined in postnatal day 13. The observed changes in pup thyroid weights and thyroxin levels are considered of no biological relevance and non-adverse due the very small size of the effects and/or lack of statistical significance and/or dose-dependency and the fact that the observed lower thyroxin levels were well within the labatory’s historical control data. Livers were not histopathologically examined in pups, but based on the observed liver findings in parental animals, it is considered likely that also in pups enzyme induction and increased hepatic clearance of thyroxin are the reason for the observed findings.

GROSS PATHOLOGICAL FINDINGS:
The visceral examination of all stillborn and died pups did not indicate any test item-related effects on the morphological development of the offspring. There were no test item-related gross observations in all pups during scheduled necropsy on PND 13. No signs of demasculinization were found in any male in all groups.

HISTOPATHOLOGICAL FINDINGS, NON-NEOPLASTIC:
No test item-related histological changes were observed in thyroid glands of male and female pups examined in postnatal day 13.
The number of pups with vacuolar changes in the follicular epithelium was slightly increased in the 100 and 400 mg/kg bw/day groups: the frequency of observation was 50% for both male and female offspring from the vehicle control group, while the maximum frequency was 82% in the 100 mg/kg bw/day male group, and 72% in the 400 mg/kg bw/day female group. However, these changes were not statistically significant compared to the control group. The cystic follicles were observed with a similar minimum frequency in all dose treated male groups. Although cystic follicles in female pups were observed only in the test item groups, the frequency of findings was minimal and was not considered to be related to the test item.
In one pup found dead on PND 0 from the 100 mg/kg bw/day dose group, the microscopic evaluation of urinary bladder with gross observation revealed fresh diffuse hemorrhage in the lamina propria of mucosa and submucosa and smooth muscle that was not assumed as treatment-related.

THYROD HORMONE ANALYSIS
The mean serum thyroxin measured in PND 4 offspring was decreased in all test item groups with a statistically significant change at 400 mg/kg bw/day compared to the value in the control vehicle group (7.4%, p < 0.05). Although historical control data on T4 serum level in the PND 4 offspring are not extensive, the mean hormone value in all test item administered groups was lower than the historical control value (MEAN ± S.D., 29.67 ± 3.63 ng/mL, Min – Max, 25.21 – 34.25 ng/mL). The level of thyroxin in the control group of PND 4 offspring was not significantly different from this value.
In PND 13 offspring, the most pronounced decrease in thyroxin level by 10.7% compared with the control group was observed in the 100 mg/kg bw/day dose group, while thyroxin levels at 400 mg/kg bw/day were not statistically different from the control. The mean values of thyroxin concentration in all test item groups were decreased compared with the mean historical control value for PND 13 offspring (MEAN ± S.D., 49.87 ± 7.96 ng/mL), but the values were within the range of historical data (Min – Max, 30.22 – 67.73 ng/mL).

The observed changes in pup thyroxin levels are considered non-adverse due the very small size of the effects and/or lack of statistical significance and/or dose-dependency and the fact that the observed lower thyroxin levels were well within the laboratory’s historical control data. Livers were not histopathologically examined in pups, but based on the observed liver findings in parental animals, it is considered likely that also in pups enzyme induction and increased hepatic clearance of thyroxin are the reasons for the observed finding.

Effect levels (F1)

1

Dose descriptor:

LOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Thyroxin hormone (T4) levels

Dose group	0 mg/kg	N	25 mg/kg	N	100 mg/kg	N	400 mg/kg	N
T4 concentration (ng/mL)	Mean ± SD		Mean ± SD		Mean ± SD		Mean ± SD
Post-natal Day 4	26.65 ± 0.82	6	24.08 ± 1.12	8	24.91 ± 1.85	8	24.67 ± 1.03 (a)	9
Post-natal Day 13	48.20 ± 4.03	8	43.98 ± 2.90	12	43.04 ± 2.36 (a)	11	45.08 ± 5.58	11
a = significantly different from control group 1 at 0.05 using ANOVA with Dunnett's test N = number of pups

Table 2: Summary of thyroid weights in F1 offsprings at post-natal Day 13

	Dose group	0 mg/kg	25 mg/kg	100 mg/kg	400 mg/kg
		n = 8/sex	n = 12/sex	n = 11/sex	n = 11/sex
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
males	Final body weight (g)	29.02 ± 3.02	29.15 ± 1.58	28.45 ± 2.72	27.60 ± 2.38
	Thyroids (g)	0.0038 ± 0.0004	0.0041 ± 0.0009	0.0039 ± 0.0007	0.0043 ± 0.0008
	Thyroids (g/ 100g body weight)	0.0131 ± 0.0016	0.0140 ± 0.0025	0.0139 ± 0.0027	0.0155 ± 0.0025 (a)
females	Final body weight (g)	28.73 ± 2.31	27.07 ± 1.72	28.10 ± 3.08	26.05 ± 2.36 (a)
	Thyroids (g)	0.0038 ± 0.0007	0.0040 ± 0.0005	0.0041 ± 0.0007	0.0040 ± 0.0008
	Thyroids (g/ 100g body weight)	0.0131 ± 0.0020	0.0149 ± 0.0017	0.0146 ± 0.0023	0.0153 ± 0.0026 (a)
a = significantly different from control group 1 at 0.05 using ANOVA with Dunnett's test

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no observed adverse effect level (NOAEL) for systemic toxicity was ≥ 400 mg/kg bw/day.
The NOAEL for reproduction was derived to be ≥ 400 mg/kg bw/day, because no adverse effects on reproduction function and performance were observed up to the highest tested dose of 400 mg/kg bw/day. The NOAEL for developmental toxicity was derived to be 100 mg/kg bw/day based on decreased pup weights on PND 13.