Morpholinium, 4-[2-[2-[(2-hydroxyphenyl)methylene]hydrazinyl]-2-oxoethyl]-4-methyl-, chloride (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, minor restriction in design, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

EC Number:

700-714-9

Cas Number:

1254469-57-2

Molecular formula:

C14H20ClN3O3

IUPAC Name:

4-({N'-[(2-hydroxyphenyl)methylidene]hydrazinecarbonyl}methyl)-4-methylmorpholin-4-ium chloride

Details on test material:

- Name of test material (as cited in study report): Tinocat ES 96000
- BASF substance number: 11/0013-2
- Molecular formula: C14H20N3O3Cl
- Molecular weight: 313.78
- Physical state: White solid
- Analytical purity: 98.4 g/100 g (Quantitative 1H-NMR spectroscopy)
- Lot/batch No.: OP 2/2011
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date: 30 November 2013

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: males 306 - 337g; females 183 - 230
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% Aq. Carboxymethyl cellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.

VEHICLE:
1% Aqueous carboxymethyl cellulose (carboxymethyl cellulose: BUFA, IJsselstein, The Netherlands; water: Elix, Millipore S.A.S., Molsheim, France).
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe.
- Dose volume: 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of dose preparations: Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

- Analysis of dose preparations: No test substance was detected in the Group 1 formulation.
The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Some females were not dosed during littering.

Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
80, 250, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study.
- Rationale for animal assignment: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Examinations

Observations and examinations performed and frequency:

PARENTAL ANIMALS
- Mortality / Viability: At least twice daily.

- Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Clinical observations (clinical signs and arena) were conducted at least immediately (0-15 min.) after dosing, based on the absence of a peak occurrence of clinical signs after dosing in the dose range finding study.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

- Functional Observations:
The following tests were performed on the selected 5 animals/sex/group:
- hearing ability *
- pupillary reflex *
- static righting reflex *
- grip strength *
* score 0 = normal/present, score 1 = abnormal/absent
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled
with warm water). The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (all before blood sampling).

- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

- Clinical laboratory investigations:
Blood samples were collected on the day of necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3- EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
Furthermore, from the selected 5 animals/sex/group an additional blood sample (1 mL) was collected into serum tubes for possible future measurement of thyroid-stimulating hormone (TSH) the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. No analysis was deemed necessary for this study. Any samples remaining at finalization of the study report were discarded.

The following parameters were determined:
Haematology:
White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets.
Clotting Potential:
Prothrombin time, Activated Partial thromboplastin time.
Clinical Biochemistry:
Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids

Sacrifice and pathology:

NECROPSY PARENTAL ANIMALS:
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
Condition Day of necropsy
Females which delivered Lactation Days 5-7.
Females which failed to deliver² Post-coitum Days 25-26 (females with evidence of mating) or approximately 21 days after the last day of the
(nos. 41, 43, 48 and 56) mating period (females without evidence of mating).
Females with total litter loss³ Within 24 hours of litter loss.
(no. 79)
Males Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females. These numbers were not reported for non-pregnant and non-mated females.

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Identification marks: not processed
Adrenal glands; (Aorta); Brain - cerebellum, mid-brain, cortex; Caecum; Cervix; Clitoral gland; Colon; Coagulation gland; Duodenum; Epididymides¹; Eyes (with optic nerve (if detectable) and Harderian gland)¹; (Male and Female³ mammary gland area); Femur including joint; Heart; Ileum; Jejunum; Kidneys; (Lacrimal gland, exorbital); (Larynx); Liver; Lung, infused with formalin; Lymph nodes - mandibular, mesenteric; (Nasopharynx); (Esophagus); Ovaries; (Pancreas); Peyer's patches [jejunum, ileum] if detectable; Pituitary gland; Preputial gland; Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach (forestomach and glandular stomach); Testes¹; Thymus; Thyroid including parathyroid if detectable; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions.

¹ Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37- 40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
² In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
³ The mammary gland area was only required for females with total litter loss in which no pups of the litter had milk visible in the stomach.

ORGAN WEIGHTS
Terminal body weight were recorded from all males and the selected 5 females/sex/group.
The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group:
Adrenal glands; Brain; Epididymides; Heart; Kidneys; Liver; Ovaries; Spleen; Testes; Thymus; Uterus (including cervix); Prostate¹; Seminal vesicles including coagulating glands¹; Thyroid including parathyroid¹
¹ weighed when fixed for at least 24 hours.
All remaining males:
Epididymides
Testes

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). In addition, Perls' Prussian Blue staining of the spleen was performed for animal nos 1, 4, 33, 34, 42, 44, 74 and 75.
Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY
A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- Based on treatment related findings in Group 4 the spleen and sternal bone marrow of selected females from Groups 2 and 3 were also examined.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals of Groups 1 and 4 and from all males that failed to sire (no. 16; 80 mg/kg bw/day) and all females that failed to deliver healthy pups (no. 56; 80 mg/kg bw/day). Male nos. 1, 3 and 8 and female nos. 41, 43 and 48 (controls) also did not have live pups, but as they were in Group 1, their reproductive organs were automatically examined. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
* Reproductive organs include the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Statistics:

The following statistical methods were used to analyze the data:
− If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
− The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
− The Fisher Exact-test was applied to frequency data.
− The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:

OBSERVATIONS PARENTAL ANIMALS
- Mortality:
No treatment related mortality occurred during the study period.
Female no. 79 (800 mg/kg bw/day) was euthanized after having a total litter loss on Day 2 of lactation. One control male (no. 3) died at blood sampling. This was in no way related to treatment.

- Clinical signs:
No clinical signs of toxicity were noted during the observation period.
Salivation was seen for all animals at 800 mg/kg bw/day and one female at 250 mg/kg bw/day. This was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to the taste or possible irritancy of the test substance.
Pale faces were noted over several days for four females at 800 mg/kg bw/day and hunched posture and piloerection were noted for a single male. At the limited incidence observed, these were not considered to be toxicologically relevant.
All remaining clinical signs were incidental in nature and included alopecia and scabbing of various body regions. These findings occurred within the range expected for rats of this age and strain and were not toxicologically relevant.

- Functional observations:
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.
The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

- Body weights:
No toxicologically relevant changes in body weights and body weight gain were noted.
Females at 800 mg/kg bw/day had significantly lower body weight gains on Day 8 of the premating period. As the difference from controls was slight, and no other effects on body weights were seen, it was not considered to be toxicologically relevant.

- Food consumption:
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Relative food consumption was significantly higher for females at 800 mg/kg bw/day from Days 14-17 of the post coitum period. The difference from controls was slight and transient, and a decrease in food consumption would be expected if treatment related toxicity were evident.

- Haematology:
There were no toxicologically relevant effects on haematology parameters up to 800 mg/kg bw/day.
At 800 mg/kg bw/day, males had significantly higher monocytes, red blood cell distribution width (RDW), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values than controls. In all cases, the changes from controls were only slight and were not accompanied by any corroborative histopathological effects. As such, they were not considered to be toxicologically relevant.

- Clinical biochemistry:
There were no toxicologically relevant effects on clinical biochemistry parameters up to 800 mg/kg bw/day.
Males at 800 mg/kg bw/day had significantly lower glucose and females at this dose level had significantly higher bile acids than controls. No toxicological relevance was attributed to these differences as the values remained within the range considered to be normal or were not supported by relevant effects on any other related parameters (bile acids).
Females at 250 mg/kg bw/day had significantly increased total bilirubin when compared to controls. In the absence of a treatment related distribution, this was not considered to be toxicologically relevant.
At 800 mg/kg bw/day the mean alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) values for males were higher than controls (not statistically significant). The differences were secondary to high values obtained for animal no. 33 and were considered to be incidental in nature.

- Macroscopic examination:
Thickened wall was noted in the duodenum and jejunum of female rats - one case in group 1 (0 mg/kg bw/day), two in group 2 (80 mg/kg bw/day), three in group 3 (250 mg/kg bw/day) and in five cases in group 4 (800 mg/kg bw/day). This finding was also noted in one case in the ileum of a group 2 (80 mg/kg bw/day) female.
The remainder of the macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls. These included pelvic dilation of the kidney, nodule on the epididymis, enlarged and/or discoloration of the liver, adrenals spleen, clitoral gland or lymph nodes, exopthalmus, foci on the thymus, lungs, stomach glandular mucosa or clitoral gland, reduced size of the liver, spleen or thymus, an accessory liver lobe, and alopecia and/or scabbing over various body areas.

- Organ weights:
Females at 800 mg/kg bw/day had significantly higher absolute (26%) and relative (29%) spleen weights than controls.
All other organ weights and organ to body weight ratios were similar between control and treated animals.

- Microscopic examination:
Microscopic findings associated with treatment were noted in the spleen and sternal bone marrow of females.
In the spleen primarily erythroid hemopoietic foci were increased in severity in the treated groups. This finding was recorded at minimal to slight degree in all five selected group 1 (0 mg/kg bw/day) animals, at minimal to moderate in all five group 2 (80 mg/kg bw/day) and group 3 (250 mg/kg bw/day) and at minimal to severe in all five selected group 4 (800 mg/kg bw/day) females. This increase in severity was not statistically significant (p > 0.05). In the sternal bone marrow erythroid hyperplasia at minimal degree was noted in one group 2 (80 mg/kg bw/day), two group 3 (250 mg/kg bw/day) and in four group 4 (800 mg/kg bw/day) females. This increase was statistically significant in group 4 (800 mg/kg bw/day) (p < 0.05) with a positive trend (p < 0.01). Positive reaction with Perls stain confirmed the nature and degree of hemosiderin deposition in the group 1 and 4 animals examined.
There were no adverse histological alterations seen in the small intestine segments noted at necropsy with thickened walls. Some segments were judged to be large but with normal histology and may have reflected physiological response to gestation and lactation.
Animal no. 79 recorded red pulp atrophy (slight) and lymphoid atrophy (slight) in the spleen and lymphoid atrophy in the thymus (moderate). These findings may indicate an underlying condition in this animal which could have contributed to the loss of litter.
In other animals suspected of infertility, there were no microscopic findings which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all animals assessed.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified