2-Propanone, reaction products with diphenylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2018 - 23 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Deviations:

yes

Remarks:

See “any other information on materials and methods incl. tables” for further details.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-Propanone, reaction products with diphenylamine

EC Number:

270-192-0

EC Name:

2-Propanone, reaction products with diphenylamine

Cas Number:

68412-48-6

Molecular formula:

Variable.

IUPAC Name:

N-cyclohexylcyclohexanamine; propan-2-one

Test material form:

solid: particulate/powder

Details on test material:

Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
Batch/Lot number: EL5D06K347
Appearance: Green to brown powder
CAS number: 68412-48-6
Purity: 100% (as a UVCB)
Retest date: 05 April 2018
Storage conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

Name: AMINOX®
CAS number: 68412-48-6
Batch/lot number: T7C16001
Description: Green to brown flakes
Purity: Considered as 100% (as a UVCB)
Expiry date: 14 March 2019
Storage condition: Controlled room temperature (15-25 °C, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI Wistar rats

Details on species / strain selection:

Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/174-220PE [4]).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany, from SPF colony.
Housing conditions: Standard laboratory conditions
Number of animals: 55 male and 55 female rats in the acclimatisation/pre-treatment period, then 48 male and 48 female rats for dosing (12 animals/sex/group for treatment, 4 groups). The additional 7 spare animals/sex were excluded during the randomisation.
Age of animals: Young adult rats, approximately 11-13 weeks old at start of treatment and 13-15weeks old at mating.
Body weight range: Males: 393 – 460 g, females: 230 – 323 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Acclimation period: 22 days
Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 239
Cage type: Type II and III polycarbonate
Bedding and nesting: LIGNOCEL® ¾ S certified wooden chips (batch number: 03018180719, expiry date: 19 July 2021) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study.
Arbocel® nest building material (batch number: 05072180528, expiry date: 28 May 2021) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding and nest building material were archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.2 – 25.3 °C (target range 22 ± 3 °C)
Relative humidity: 23 – 60 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.
Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 840 33675, expiry date: 31 January 2019 and batch
number: 639 38520, expiry date: 30 April 2019), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificates for the batches used, which are archived with the raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are archived with the raw data at Citoxlab Hungary Ltd.

Animal identification
Each adult/parental animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at Citoxlab Hungary Ltd.
During the pre-exposure period, animals were identified with temporary numbers only. After this 2-week period, a randomisation was performed based on the body weights and the selected animals received their final animal numbers, as follows:
This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.
Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth.
Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.
Dosing of both sexes began, after 22 days of acclimatisation and pre-exposure period (14 days), 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, on 25 days after the day of presumed mating.
All F1 offspring were terminated on Day 13 post-partum (F1 offspring selected for blood sampling in PND4 were terminated on that day). In order to allow for overnight fasting of dams with urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC-UV

Details on analytical verification of doses or concentrations:

SOLUTIONS, MIXTURES
Vehicle: 1,2-Propylene glycol (PG) was the Vehicle.
Diluent: Tetrahydrofuran:acetonitrile 1:9 (V/V) was used as Diluent.
Stock Solution: Approx. 100 mg test item was weighted with 0.01 mg precision into 10 mL volumetric flask and filled up with tetrahydrofuran. The solution was mixed well by hand. The concentrations of the test item was approx. 10 mg/mL.
Working Solution: The concentration of the working solution was 1000 μg/mL.
Calculated amount of Stock Solution was pipetted into 10 mL volumetric flask and filled up to volume with acetonitrile. The solution was mixed well by hand.
Calibration Solutions: The Working Solution was diluted to the 100 – 1000 μg/mL range with Diluent.
The calibration solutions were injected in duplicates.
ANALYTICAL SAMPLE PREPARATION
0.5 mL samples were received from the top, middle and bottom of the formulation vessels and the samples were diluted into the dynamic range of the calibration in one or two steps. All samples were injected in duplicates.
Preparation of Control and 20 mg/mL formulations: 0.5 mL aliquots (measured with 0.1 mg precision) was diluted to 20 mL with Diluent.
Preparation of 60 mg/mL formulation: 0.5 mL aliquots (measured with 0.1 mg precision) was diluted to 20 mL with Diluent.
5000 μL from the intermediate dilution was pipetted into 10 mL volumetric flask and filled up with Diluent.
Preparation of 200 mg/mL formulation: 0.5 mL aliquots (measured with 0.1 mg precision) was diluted to 20 mL with tetrahydrofuran. 1000 μL from the intermediate dilution was pipetted into 10 mL volumetric flask and filled up with acetonitrile.

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/postmating
periods. This was 28 days in total for males. Females were treated throughout gestation and
up to and including postpartum/lactation day (PPD) 13.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five animals per sex per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and
females were randomised separately.

Rationale for dose selection and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 over 2000 mg/kg bw in rats [3]) and results of the Dose Range Finding (DRF) study performed at the Test Facility (Citoxlab study code: 17/174-220PE [4]), where no adverse effects were seen up to 1000 mg/kg bw/day.

The aim of this study was to use a maximum of 1000 mg/kg bw/day or to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from the preliminary study, doses of 100, 300 and
1000 mg/kg bw/day were selected for the main study.
The oral route was selected as it is one of the possible routes of human exposure.

Positive control:

Not specified

Examinations

Observations and examinations performed and frequency:

Clinical observations and functional observation battery (FOB)
All animals:
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily*, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. The principles and criteria summarized in the Humane Endpoints Guidance Document (OECD) were taken into
consideration [6].
More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respirato
ry pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
*Note: No general clinical observations were made on those days when detailed clinical observations were made.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 25; females on PPD9-12). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during SMART
measurement.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table.
The distance between the two resulting ink spots for the hind limbs was measured.
Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on
each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed [7].
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed condition s. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g at least weekly during the pre-exposure period, then on Day 0 for randomisation purposes, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1g weekly (on the days of body weight measurements).

Sacrifice and pathology:

All animals were euthanized under pentobarbital anaesthesia by exsanguination. After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were
observed macroscopically.

Gross findings
Lungs with bronchi 8
Skeletal muscle (quadriceps)
Adrenals
Lymph node 9
Small intestine 10
Animal identification 1
Ovary
Spinal cord 11
Aorta 2
Oviduct
Spleen
Brain 3
Pancreas
Sternum with marrow
Epididymis
Pituitary
Stomach
Eye with the optic nerve 4
Prostate
Testis
Oesophagus
Salivary gland (including mandibular, sublingual and parotid glands)
Thymus
Femur with marrow
Thyroid with parathyroid gland 4
Heart 5
Tongue
Kidney
Sciatic nerve
Trachea
Large intestine 6
Seminal vesicle with coagulating gland
Urinary bladder
Extraorbital lachrymal gland
Uterus 12
Harderian gland
Skin, subcutis with mammary gland (inguinal)
Vagina
Liver7
 
 
 
1. Fixation and preservation only.
2. Aorta thoracic and abdominal.
3. Section according to the STP recommendations.
4. If applicable, parathyroids and optic nerves were examined histologically only if present in routine sections.
5. Section including both ventricles and atria, septum with papillary muscle.
6. Caecum, colon and rectum.
7. Liver, 3 lobes, left lateral, right medial, caudate.
8. Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal.
9. Mandibular and mesenteric.
10. Duodenum, ileum and jejunum with Peyer’s patches.
11. Transverse sections, 3 levels – cervical, thoracic and lumbar.
12. Horns, body and cervix.
The eyes with the optic nerve, testes and epididymides were retained in modified
Davidson’s fixative. All other organs were retained in 10% buffered formalin solution.
No histopathology evaluation was performed. After finalization of the Report, the
preserved organs and tissues will be discarded.

Statistics:

Group means and standard deviations were calculated from numerical data obtained in the study. The statistical evaluation of appropriate data (marked as † in the list below) was performed with the statistical program package of SAS 9.2 (when using Provantis) or with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups will be checked by Bartlett's test. Where no significant heterogeneity is detected, a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant, then Duncan's Multiple Range test is used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data is examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance is applied. If a positive result is detected, the inter-group comparisons are performed using Mann-Whitney Utest.
The Chi-squared test will be used for non-continuous data In case of the SAS 9.2 software package (within the validated Provantis system) the following
decision tree is applied automatically for statistical evaluation of continuous numeric data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Piloerection, hunched back, decreased activity were seen in the found dead animal (#4511) before its death. As the animal died because of a gavage accident, these symptoms are considered not to be test item related.
Piloerection was seen in all other High dose females from Day 35, which disappeared in all animals until Day 41. The High dose animals littered between Day 36 and 40, so this clinical sign was observed around the parturition.
All male animals were symptom-free during the study.
Piloerection, hunched back and decreased activity were seen in the pre-terminally euthanized animal (#1509) before its euthanasia. As the animal was euthanized because of a parturition problem, these symptoms are considered not to be test item related.
The following individual observations were not attributed to systemic effects of the test item: Tonic convulsion in one Control female (#1510) on Day 42, in two Mid dose females (#3501, 3505) on several occasions from Day 41, in one High dose female (#4503) on Day 43, slight noisy respiration in one Low dose female (#2509) on Day 1 and in on Mid dose female (#3512) on Days 10-11, alopecia on all limbs of one High dose female (#4512) from Day 40.
Green discoloured urine was seen in one High dose female (#4505) during the last 3 days before terminal necropsy, the observation was of uncertain relationship with treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related mortality was seen during the study.
One High dose female (#4511) was found dead on Day 19. The cause of death was confirmed at necropsy as a gavage accident.
One Control female (#1509) was pre-terminally euthanized due to a problematical parturition (on the 24th day of gestation, Day 40 of dosing), because of animal welfare reasons. The animal gave birth to 6 dead pups and was not able to complete the parturition. Before the euthanasia, blood was seen at the urogenital area besides the other clinical symptoms.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, significantly lower body weight gain was observed during the first week of treatment in the Mid and High dose groups. After this, the bodyweights were relatively compensated and all male dose groups had similar body weight as the controls at the end of the observation period.
In all female dose groups, body weight loss was observed during the first week of treatment, and lower body weight gains compared to the controls during the second week (pre-mating period). After that, during the gestation period, the test item treated females started to gain weight normally, but still slightly below the control means. After the parturition, during the lactation period, the female body weight gain values became normal, however as a consequence of the earlier lower body weight gains, the
female animals’ body weights did not catch up to the control values.
Overall, in both sexes, the first week of treatment was the most significant effect: The Mid and High dose males had reduced body weight gain and the Low, Mid and High dose females had body weight loss. In both sexes, after 1 or 2 weeks of treatment, the body weight gain in all dose groups were near parallel to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, there were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls. The measured values were within the normal range for this strain and age.
The food consumption of the females showed similar pattern as the body weight changes; there were statistically significantly lower food consumptions in all dose groups during the pre-mating period, slightly lower food consumption during the gestation period without attaining statistical significance, and generally normal food consumption during the lactation period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were within the historical control ranges. These differences were considered to not reflect an effect of the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No other clear item related clinical chemistry findings were seen. All significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an effect of the test item.
Thyroid hormone levels (adult males): Significantly lower (p<0.05) T4 value was recorded in the High dose group, which was most probably incidental, not related to the test item. As all individual values in the High dose group were well within the historical control range, therefore this difference is not considered toxicologically relevant.

It is considered that there was no evidence that test item had a direct effect on thyroid hormone levels.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No clear other test item-related changes were observed in the urinalysis parameters. The significant differences were considered to be incidental, not reflecting an effect of the test item.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of grip strength, foot splay or locomotoractivity. A statistically significantly lower hind limb foot splay mean in the Mid dose group is considered to be incidental, not test item-related. The High dose group had very similar value to the Control group.
All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on organ weights.
Statistically significantly (p<0.05) higher relative (to body) liver weights were seen in the male High dose group, which correlated with the histopathological findings.
All other statistically significant values were considered to be incidental and not test item-related. These observed values were within the historical control ranges in all cases and there were no clear dose responses observed in these parameters, thus these statistical differences were considered to have no toxicological significance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following findings were seen in the found dead animal (#4511): Yellow mucoid material in the cecum, jejunum and stomach, 1 x 7 mm cervical peroration of the oesophagus, approx. 3 mL brownish creamy material in the thoracic cavity, pale diffuse bilateral discoloration of the kidneys and dark red diffuse discoloration of all lobes of the non-collapsed lungs.
The oesophagus perforation and the presence of presumably test item in the thoracic cavity are considered as clear signs of a gavage accident. The other findings are considered as agonal or postmortem changes.
The following findings were seen in the preterminally euthanized animal (#1509): Red liquid on the fur at the urogenital area, bilateral dilatation of the uterine, pale diffuse discoloration of all lobes of the liver, pale diffuse discoloration of the pancreas, many pale foci in the glandular mucosa of the stomach, bilateral diffuse pale discoloration of the thyroid glands.
The animal was euthanized because of a problematical parturition issue. All observed findings at necropsy are considered to be related to the problematical parturition of the animal and consequential changes before termination.
No test item-related macroscopic changes were observed in the terminally euthanized animals. The following incidental or background findings were seen in the terminally euthanized animals: unilateral pelvic dilatation of the kidney (2002, 4003), red discoloration of the thymus (#2004), unilateral small thyroid gland (#2501), unilateral enlargement of testis (#3012, 4011), unilateral red diffuse discoloration of ovary, thin fur on all limbs (4512 The bilateral dilatation of the uterine, seen in 3 females (#4509, 2504, 2510), is a normal sign of oestrus in rats.
The black liquid seen in the uterine and vagina of one High dose animal (#4502) was a sign of abortion, as was confirmed at the counting of implantation sites and corpora lutea. The animal did not give birth to any pups. This was considered to be a background finding, unrelated to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were seen in the liver of the High dose animals: Minimal centrilobular hypertrophy in 5/5 High dose males and in 1/5 High dose females, minimal to mild hepatocellular vacuolation in 3/5 High dose males. These liver changes at the High dose were considered to be minor treatment related, non-adverse findings.
Besides these observations, based on the low incidence and/or severity and/or distribution crosscontrol and dosed animals, all other observations were considered to be incidental or a common background.

Moderate multifocal degeneration/necrosis of the cardiac muscle cells, severe bilateral chronic progressive nephropathy, mild hepatocellular vacuolation, mild cholangiofibrosis and mild focal/multifocal hepatocellular necrosis in all lobes of the liver and moderate decreased cellularity in the cortex of thymus were recorded in the found dead animal.
Mild focal/multifocal hepatocellular necrosis in all lobes of the liver, minimal focal necrosis in the glandular mucosa of the stomach and mild decreased cellularity in the cortex of the thymus were recorded in the preterminally euthanized animal. These changes were ascribed to the problematical parturition, unrelated to treatment.

Gravidity was confirmed in the uterine.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating indices were 100% in all groups. The male and female fertility indices were 92% in the Control, 50% in the Low dose group, 67% in the Mid dose group and 92% in the High dose group. Although the number of non-pregnant females in
the Low and Mid dose groups were higher than expected, the High dose group had exactly the same incidence as the Control group, therefore it can be concluded that the test item did not have an effect on the reproductive parameters.
Test item administration was considered to have no impact on the duration of the mating period.
Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 3 days of pairing (cohabitation) for all females.
The mean duration of mating was 2.0, 2.3, 2.7 and 2.8 days in the Control, Low, Mid and High dose groups, respectively.
There was no effect of treatment noted during the gestation period in any of dose groups. The higher pre-natal mortality in the High dose group was caused by one animal (#4502), which lost all of its pups during gestation. Excluding this animal’s data, the mean of the pre-natal mortality in the High dose group would be below the Control. This individual animal’s loss of pups is within the expected range and is not attributed to treatment.
The statistically significantly lower number of implantation sites in the Low dose group was caused by the low number of pregnant animals (non-pregnants are zero values in the mean), and not by lower number of implantation sites in the pregnant animals. The group mean without the non-pregnant animals can be seen in brackets.
The mean duration of pregnancy was comparable in the Control and test item treated groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In summary, daily administration of AMINOX® by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in test item related mortality.

Transient piloerection was seen in the High dose females around the parturition. No changes at neurological assessment were seen at the end of the treatment period.

During the first 1-2 weeks of treatment, the Mid and High dose males had reduced body weight gain and the Low, Mid and High dose females had body weight loss. After that, the body weight gain in all male and female dose groups were near parallel to controls.

At clinical pathology, there were no test item-related findings.

At necropsy, no test item related macroscopic changes were seen. Slightly higher liver weights were recorded in the male High dose group. At histopathology, hepatocellular hypertrophy and vacuolation were seen in the male High dose group. These findings were considered, adaptive, non-adverse changes.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs or at observations following euthanasia.

No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

The NOAEL for Systemic toxicity for the adults was considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of the test item AMINOX® following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive/ developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The Experimental Design was as follows:

Group Number	Group designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	0	0	5	1001-1012	1501-1512
2	Low Dose	100	20		2001-2012	2501-2512
3	Mid Dose	300	60		3001-3012	3501-3512
4	High Dose	1000	200		4001-4012	4501-4512

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily and/or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

RESULTS 

In summary, daily administration of AMINOX®by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day, under the conditions of this study, did not result in test item related mortality.

Transient piloerection was seen in the High dose females around the parturition. No changes at neurological assessment were seen at the end of the treatment period.

During the first 1-2 weeks of treatment, the Mid and High dose males had reduced body weight gain and the Low, Mid and High dose females had body weight loss. After that, the body weight gain in all male and female dose groups were near parallel to controls.

 

At clinical pathology, there were no test item-related findings.

At necropsy, no test item related macroscopic changes were seen. Slightly higher liver weights were recorded in the male High dose group. At histopathology, hepatocellular hypertrophy and vacuolation were seen in the male High dose group. These findings were considered, adaptive, non-adverse changes. 

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs or at observations following euthanasia. 

No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 1000 mg/kg bw/day.

The NOAEL for Systemic toxicity for the adults was considered to be 1000 mg/kg bw/day.