Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2017 - November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

EC Number:

947-738-8

Molecular formula:

Molecular formulas of exemplary components: C33H68KO10P C49H100KO16P C24H49K2O10P C24H50KO10P C16H34KO4P C8H17K2O4P C8H18KO4P

IUPAC Name:

Phosphoric acid, mixed esters with alcohols, C7-9-iso-, C8-rich and alcohols, C11-14-iso-, C13-rich, ethoxylated, potassium salt

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal age at the start of the treatment period: approx. 14-15 weeks old. Females were nulliparous and non-pregnant at the start of the study.
Body weight at the allocation of the animals to the experimental groups: males 338-370 g (mean 355.73 g); females 226-253 g (mean 240.20 g)

Housing and feeding:
Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals will be housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females will be housed in groups of 2 in tpe III IV polysulphone cages and during post-mating period for males depending on the mating status. During mating period males and females will be housed together in ratio 1:1 (male to female). After the confirmation of mating, females will be kept individually during gestation/lactation period in type III H, polysulphone cages and males will be returned to their original cage. In each cage Altromin saw fibre will be used as bedding.
- Nesting material provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua ad injectionem, Delta Medica

Details on oral exposure:

The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 10 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

On days 1 and 2, due to technical error half of female animals were dosed with 5 mL/kg. For compensation the pre-mating period of female animals was extended from 14 to 15 days.

Concentrations in vehicle:
LD 13.34 mg/mL test item (12 mg/mL active ingredient)
MD 33.24 mg/mL test item (30 mg/mL active ingredient)
HD 83.33 mg/mL test item (75 mg/mL active ingredient)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
According to the study the test item was homogenous (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples) were taken.
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were stored at < -20 °C before they were shipped and analysed. The B-samples were retained at -15 to -35 °C at test facility and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle for 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating for males and 15 days of pre-mating for females and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period for 29 days.

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

per active ingredient (90%, conversion factor 1.1)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

per active ingredient (90%, conversion factor 1.1)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

per active ingredient (90%, conversion factor 1.1)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were selected based on previous dose range finding study. The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Inadvertently, they were not performed in week 5 on male animals.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, PND 9 and PND 13 along with pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION: Yes
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

OPHTHALMOSCOPIC EXAMINATION: Yes
In the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group.

HAEMATOLOGY: Yes
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined:
haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH),
mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc)

Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined:
prothrombin time (PT), activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K)
From 2 preferably female pups / litter on day 4 after birth from all dams and 2 pups / litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations, except for dam no. 76, 47 where one female and one male pup were taken. No pups were eliminated as litter size dropped below 8 pups. As there was only one pup available above a litter size of 8, only one pup was sacrificed.

URINALYSIS: Yes
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
The following parameters were measured using qualitative indicators:
specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones) , urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature.

Sacrifice and pathology:

All surviving males were sacrificed on study day 30 (a day after the last dosing) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine).
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.

Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral- buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.

Organ weights
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals euthanised for animal welfare reasons were not recorded.
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Organs weighed at necropsy:
testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), kidneys (paired weight), adrenal (paired weight), pituitary gland, uterus with cervix, ovaries (paired weight), thymus, liver, spleen, brain, heart.

The following tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol:
adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, femur with knee joint (no hist-path), Harderian glands (no hist-path), heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (axillary, no hist-path), lymph nodes (mandibular), lymph nodes (mesenteric), mammary gland area (male and female, no hist-path), oesophagus (no hist-path), optic nerves (no hist-path), ovaries, oviducts (no hist-path), pancreas (no hist-path), pituitary (no hist-path), prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular, no hist-path), sciatic nerve, skeletal muscle, skin (no hist-path), spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue (no hist-path), trachea, ureters (no hist-path), urinary bladder, uterus with cervix and vagina, head (no hist-path), nasal cavities (no hist-path).

All animals intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.

A full histopathology was carried out on the preserved organs and tissues mentioned above (except where it has been otherwise stated) of all selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for stomach, liver, adrenal glands, thymus and lung of male and female animals and kidneys of male animals as treatment-related changes were observed in the high dose group.
A full histopathology was carried out on the preserved organs and tissues of all animals which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Immunohistochemistry of α2µ-globulin was performed on kidney of male animals sacrificed at the end of the treatment period of the study (21 animals, 5 randomly selected male).

Immunohistochemistry was performed with Rat α2µ-Globulin Antibody. The specific staining was performed with e.g. Chromotrope-Aniline Blue (details to be reported). The image analysis was performed by the CELL Analysis System. Pictures of kidney slides were taken by an Olympus UC30 camera at a magnification of x20. The positive area on the total area was measured in mm2 and calculated as % on the total area. The relative values of positive structures (α2µ-globulin) were used for descriptive statistics (mean, standard deviation, minimum, maximum).

The histological processing of tissues to microscope slides and histopathological evaluation were performed at the GLP-certified contract laboratory AnaPath GmbH, Switzerland.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.
The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, clinical abnormalities, body weight changes, effects on mortality and any other toxic effects.
Parameters like body weight gain and food consumption were calculated as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.

A statistical assessment of the results of body weight and food consumption was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals sacrificed for animal welfare reasons:
On PMD 15 LD group female was seen with moderate piloerection, moderate sunken flanks, abnormal breathing, pale skin and hypothermia and was euthanised.
MD group female was sacrificed in a moribund condition on GD 20, showing moderate piloerection, moderately reduced spontaneous activity. HD female was seen with severe abnormal breathing, moderate piloerection, severe increased salivation and muscular hypotonia on PMD 13.
HD male was sacrificed on PND 4 in a moribund condition with moderate piloerection, reduced spontaneous activity, half eyelid-closure, abnormal breathing, nasal discharge and moderate salivation.

All observations (including animals euthanised for welfare reasons):
Piloerection was noted on different study days for 1/10 LD group females, 5/10 MD females and 2/10 HD females; as well as 2/10 HD males.
Nasal discharge was seen in 2 HD group males.
Abnormal breathing before application was noted on different study days in 2/10 LD females, 1/10 MD female. 1/10 LD group, 4/10 MD group and 2/10 HD group females and 1/10 LD group males and 1/10 HD group males were seen with abnormal breathing after application on different study days.
On different study days 1/10 LD group females, 4/10 MD group females and 1/10 HD group females regurgitated test item. All animals recovered afterwards except for female which was euthanized due to animal welfare reasons 3 days after regurgitation.
On several study days, female animals of the control, LD, MD and HD group and males of the LD, MD and HD group were observed moving the bedding with the nose or/and salivating immediately after administration. These signs are not assumed to be a sign of systemic toxicity.
Two females of the control group and one female of the MD group had alopecia on different study days and during different time periods. Alopecia was also noted in 1/10 LD group and 1/10 MD group males on different study dates.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One HD male was sacrificed on premating day 4 moribund for animal welfare reasons. LD female was sacrificed moribund on the last day of premating (PMD 15). MD female was sacrificed moribund on GD 20. HD female was sacrificed in a moribund condition on PMD 13.
All animals showed lung affections deemed to be due to accidental aspiration of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout the study the mean body weight of the male animals of the HD group was slightly lower than in the control (5.7 % lower than control at the end of treatment). Overall mean body weight gain was slightly and significantly (p<0.01) lower in the HD group but with high inter individual differences; as 3 out of 9 HD males lost weight and the others (6 animals) gained weight during the study. This is considered test item related.

The mean weight of the HD females during the treatment period was slightly higher than seen in the controls gaining significance (p<0.01) at gestation days 0 and 14. As body weight gain was in the normal range of biological variation this effect is not considered to be adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A tendency towards lower food consumption was seen in male and female animals of the HD group during the first pre-mating week. This is not considered toxicologically relevant. Besides, there were no considerable differences in food intake between dose groups and control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean neutrophil rate of male animals in the LD, MD and HD were slightly but dose dependently higher than control. The mean values of the HD male animals were statistically significantly (p<0.01) higher than seen in the control group (24.3 % vs. 12.4 % in the control group). In the HD group this is based on higher neutrophil rate observed in two animals (no. 37 and no. 39), indicating a slight inflammatory process. This correlated with a slight but statistically significantly lower rate of lymphocytes in the HD group (72.4 % vs. 84.3 % in the control group).
This tendency towards a higher neutrophil rate can also be seen in female animals.

A slight but statistically significantly lower mean haemoglobin and haematocrit was observed in male but not female animals of the HD group (14.7 % vs. 16.2 % and 45.4 % vs. 49.6 % in the control group, respectively). Slightly less prominently this was was also observed in males of the MD group (15.1 % vs. 16.2 % and 46.0 % vs. 49.6 % in the control group, respectively). At the same time only a tendency towards lower RBC count was seen. Individual parameters were well in the range of biological variation and without any clinical or histological association this is not considered to be test item related.

Slightly but statistically significantly higher mean platelet count of the LD and HD group male animals is not considered biologically relevant. In the absence of dose- dependency this is also not assumed to be test item related.

Besides, there were no considerable differences in haematology parameters between dose groups and control group.

Treatment with the test item had no effect on blood coagulation parameters at the end of the study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean cholesterol values of the HD group males and females and MD group males were slightly but statistically significantly lower than in controls. Due to its slight degree this decrease is not considered to be toxicologically relevant.

Slight but statistically significantly higher mean total protein and albumin values in males of the LD group are not considered toxicologically relevant. Without any dose dependency and histological correlation this is deemed to be incidental.

Liver markers of hepatotoxicity (ALAT and ASAT) were increased in female animal no. 74 of the HD group. This was an isolated finding as liver parameters were normal in the remaining male and female animals of this group.

Besides, there were no considerable differences in clinical biochemistry between dose groups and control group.

Urinalysis findings:

no effects observed

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):

The mean absolute liver weight of the MD group (25 % higher than control) and HD group (36 % higher than control) males was statistically significant higher than the control. This was also the case with normalized liver weight (to body weight) of LD males (15 %), MD males (24 %) and HD males (40 %) when compared to control. The female mean and relative liver weight of the HD group (22 % higher than control) was also significant higher than in the control. This correlated to hepatocellular hypertrophy observed histopathologically.

Normalized kidney weight was statistically significantly higher in male animals of the HD group when compared to controls (13 % above controls). This correlated histopathologically with hyaline inclusions and increased incidence of tubular basophilia. A non-significant weight increase was also found in male animals of the MD group (8 % above controls).

Moderately, however, not statistically significantly higher adrenal gland and thymus weight was observed in female animals of the LD, MD and HD groups. This correlated histopathologically with stress-related findings of minimal adrenal cortical diffuse hypertrophy (zona fasciculata) in MD and HD groups and increased incidence and severity of thymus atrophy in HD group.

The female mean ovarian weight of the HD group was slightly but significant higher in absolute weight (23 % above controls) and when normalized to body weight (21 % above controls). This was not correlated with microscopic changes or with a considerable change in fertility and is not considered toxicologically relevant.

Besides, there were no considerable differences in organ weight between dose groups and control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The animals sacrificed in moribund condition during the study had following macroscopic findings: HD male was seen with gas filled stomach jejunum and ileum. LD female and MD female showed an abnormal colored lung (white). LD and HD females showed failure to collapse of the lung. MD female showed gas filled with duodenum, jejunum, ileum and colon.

At terminal sacrifice male animal of the HD group had pelvic dilation. Moreover irregular surface was observed in its forestomach, which was also seen in other 2 male animals of this group.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Decedents:
In the lungs from all animals, there was an alveolar foreign material indicating test item aspiration. In the lungs from animal from group 2, 3 and 4 there was a chronic multifocal interstitial inflammation. In addition, in the lungs of HD animal, there was granuloma formation. In addition, the forestomach was affected by hyperkeratosis and epithelial hyperplasia and/or inflammation in HD animals. Furthermore, a slight hepatocellular necrosis in the liver from HD male was a contributing factor to poor condition.

Stomach:
An irregular forestomach in three group 4 males was deemed to be test item-related and correlated to inflammatory forestomach lesions. The histological correlate was characterized by epithelial hyperplasia, hyperkeratosis and/or inflammation in animals from all test item-treated groups, whereby in the male at 120 mg/kg, there was only one case of a minimal hyperkeratosis. The latter is considered a lesion within the range of spontaneous background alterations due to its isolated appearance. Furthermore, there were single cases of forestomach erosion in animals at 300 and 750 mg/kg.

Liver:
Hepatocellular hypertrophy, (mainly centrilobular), was noted in all test item-treated groups with a dose-dependent increase in incidence. In one decedent male at 750 mg/kg, there was a slight focal hepatocellular necrosis.

Kidney:
Hyaline inclusions in tubular cells of males increased in severity mainly at 750 mg/kg. At this dose, there was also a case of minimal granular casts, indicating tubular cell necrosis. Tubular basophilia did not increase by dose, and the incidences are considered to be within the range of control alterations. The immunohistochemical evaluation revealed statistically significantly increased values for α2-microglobulin for all test item-treated groups with highest values in group 4.

Adrenal Gland:
In females at 120 and 300 mg/kg, and in both sexes at 750 mg/kg, there was a diffuse cortical hypertrophy.

Thymus:
Thymus atrophy increased in incidence and severity in test item-treated groups.

Peyer’s Patches:
In single animals at 750 mg/kg, there was a minor atrophy of the Peyer’s patches.

Other Findings:
There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change.
Female of group 3 was not pregnant. In this animal, but also in its mating partner male, there were no abnormalities in the reproductive organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test in male and female Wistar rats with dose levels of 120, 300, and 750 mg/kg body weight day the following conclusions can be made:

Four animals were sacrificed during the study due to poor condition, including one female each from groups 2, 3, and 4, and one male from group 4. All animals showed lung affections deemed to be due to accidental aspiration of the test item (alveolar foreign material, and/or chronic multifocal interstitial inflammation and/or granuloma). In addition, the forestomach was affected by hyperkeratosis and/or epithelial hyperplasia and/or inflammation in both high dose animals. Furthermore, a slight hepatocellular necrosis in the liver from the high dose male was a contributing factor to poor condition.

An irregular forestomach in three group 4 males was deemed to be test item-related and correlated to inflammatory forestomach lesions. The histological correlate was characterized by epithelial hyperplasia, hyperkeratosis and/or inflammation in animals at 300 and 750 mg/kg. Furthermore, there were single cases of forestomach erosion in animals at 300 and 750 mg/kg. These findings are deemed to represent an irritative potential of the test item. This local finding is not considered to be relevant for human due to the restriction to the forestomach.

Hepatocellular hypertrophy, (mainly centrilobular), was noted in all test item-treated groups with a dose-dependent increase in incidence. In one decedent male at 750 mg/kg, there was a slight focal hepatocellular necrosis. If the latter finding of necrosis is a primary effect caused by the test item, remains unclear. At 300 mg/kg, no adverse finding, e.g. liver cell necrosis, accompanied the hepatocellular hypertrophy. Therefore, this dose is deemed to be the NOAEL.

In kidneys, hyaline inclusions in tubular cells of males increased in severity mainly at 750 mg/kg. The immunohistochemical evaluation revealed statistically significantly increased values for α2-microglobulin for all test item-treated groups with highest values in group 4. At 750 mg/kg, there was also a case of minimal granular casts, indicating tubular cell necrosis. Summarized, the renal alterations are indicative of α2- microglobulin-related nephropathy that is a specific male rat renal diseases and not relevant for human although of adverse character at the high dose.

Indicators of stress consisted of cortical adrenal hypertrophy in animals from all dose groups, atrophy of Peyer’s patches in single animals at 750 mg/kg, and increasing thymus atrophy in all test item-treated groups. These findings are considered as secondary test item related effects related to forestomach lesions.

Based on the pathology evaluation and the finding of necrosis in the liver of one high dose male as well as the granular casts indicating tubular cell necrosis in the kidneys of one high dose male, the NOAEL for general toxicity may be established at 300 mg/kg.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

Organ:

kidney

liver

Applicant's summary and conclusion

Conclusions:

Based on the study the NOAEL for general toxicity may be established at 300 mg/kg.

Executive summary:

Based on the study the NOAEL for general toxicity may be established at 300 mg/kg.