[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2017 to 26 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-368, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

EC Number:

275-604-2

EC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Cas Number:

71550-24-8

Molecular formula:

C24H30N3O2.Cl

IUPAC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Test material form:

liquid

Details on test material:

Storage : -15 to +40 °C
Form: yellow liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 14-15 weeks
- Weight at study initiation: males: 356 – 475 g; females: 218 - 266 g
- Fasting period before study: none
- Housing:
5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males During mating period males and females were housed together in ratio 1:1 (male to female).
After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55±10%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item.
The test item formulations were prepared at least once every 10 days (within stability time frame as given by Eurofins Munich Study No. 170528). The prepared formulation was stored at room temperature. Formulates were kept under magnetic stirring during the daily administration.

- VEHICLE : The vehicle was selected based on the test item’s characteristics and testing guideline
Name: PEG 400
Batch No.:S7263585646 / S7378385710
Physical State: liquid
Storage Conditions:at room temperature
Expiry Date:17 August 2018

Dosing volume: 4 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC Conditions
Column: XBridge, C18, 3.5 µm, 1 x 50 mm; (Art. Nr: 186003021, Waters z.B. BSL Säule Nr. 32)
Precolumn: C18, 4 x 2.0 mm, Art.Nr. AJ0-4286, Phenomenex
Injection volume: 15 µL
Sample storage temperature: 22°C
Oven temperature: 40°C
Flow rate: 0.5 mL/min
Solvent A: 3.8539 g/L Ammoniumacetate in water;Solvent B: Acetonitrile
Gradient: Time [min] A [%] B [%]
0.00 85 15
10.00 5 95
11.90 5 95
12.00 85 15
16.00 85 15
Run time: 16 min
Retention time: Approx. 9.15 min, main component
Detection: 254 nm

Calibration: 0.01-0.52 mg/mL linear r= 1.000 (accuracy 99.5-100.8% of spiked)
QC samples (25.53, 75.13 and 154.1 mg/mL): 94.9-99.8% of nominal (COV 0.63-1.48%)
Stability ((25.53 and 154.1 mg/mL):: 93.4-105.7% and 98.8-101.4% (stability measured over 6 hours, RT; 10 days, RT; 10 days, 2-8°C ;10 days, -15 to -35°C)
Homogeneity ((25.53 and 154.1 mg/mL): 94.0% (COV 0.2%) and 98.1% (COV 0.2%)

Accuracy of concentrations applied

Dose group Study week Sample no. Nominal concentration mg/mL Measured concentration
mg/mL Recovery (%) Mean Recovery (%) COV (%)
C 1 1 0 0.0 NA NA NA
2 5 0.0 NA
3 9 0.0 NA
last 13 0.0 NA
LD 1 2 25 23.55 94.2 96.4 2.3
2 6 23.74 95.0
3 10 24.66 98.6
last 14 24.47 97.9
MD 1 3 75 70.45 93.9 95.7 2.4
2 7 70.36 93.8
3 11 73.92 98.6
last 15 72.24 96.3
HD 1 4 150 145.9 97.2 98.3 1.8
2 8 146.7 97.8
3 12 151.3 100.9
last 16 145.8 97.2

Wk 1 QC samples during analytical measurements (26.48, 75.19 and 150.2 mg/mL): 94.6-97.3% of nominal (COV 0.06 - 0.42%)
Wk 3 QC samples during analytical measurements (26.35, 74.62 and 150.4 mg/mL): 95.1-96.4% of nominal (COV 0.62 - 0.87%)
Wk 5 QC samples during analytical measurements (27.28, 75.29 and 150.2 mg/mL): 98.2-99.8% of nominal (COV 0.13 - 0.33%)
Last Wk QC samples during analytical measurements (27.90, 77.34 and 150.1 mg/mL): 96.4-99.9% of nominal (COV 0.09 - 1.78%)

Duration of treatment / exposure:

females maximum exposure of 63 days in total (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12).
males minimum of two weeks prior to mating, during the mating period and up to two weeks post-mating

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on a dose range finding study (see below) and a 90-day toxicity study (Bayer 1984, NOAEL 84 mg/kg bw)

On the basis of this dose range finding reproduction/ developmental toxicity screening test with C.I. Basic Yellow 90 in male and female Wistar rats with dose levels of 50, 150, and 300 mg/kg body weight day the following conclusions can be made:
There were no adverse effects observed on body weight and food consumption in male and female adult animals. There were no adverse clinical symptoms observed during the treatment period. At necropsy, there were no macroscopic findings and no effects on organ weights.
The litter parameters including the number of pup births, no. of live pups, male and female pups, sex ratio, total litter weight, male litter and female litter weight were not affected. The pre- and post-natal parameters including the number of corpora lutea and the number of implantation sites were not affected. There were no treatment-related increases in the percentage of pre- and post-implantation losses.
Based on the generated data the dose levels 100, 300 and 600 were considered for the main OECD 422 study with C.I. Basic Yellow 90. In this conclusion the mortality seen in a 90-day study on the substance (Bayer 1984) in males is also taken into account.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: clinical observations at least once a daily, mortality twice daily

DETAILED CLINICAL OBSERVATIONS: Yes in a standard arena
- Time schedule: weekly starting pre-test: 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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly in males and females premating; females on (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: weekly in males and females premating; females on (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes as part of the functional observations pre-treatment and in the last week of the treatment for 5 males and 5 (lactating) females/dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the last week of the treatment for 5 males and 4 (lactating) females/dose from abdominal aorta
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified
- Parameters checked: 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), prothrombin time (PT) and activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the last week of the treatment for 5 males and 4 (lactating) females/dose from abdominal aorta
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified;
- Parameters checked: 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)

URINALYSIS: Yes
- Time schedule for collection: in the last week of the treatment for 5 males and 5 (lactating) females/dose qualitatively with Henry Schein Urine Stripes
- Animals fasted: Not specified
- Parameters checked:color, appearance, specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in the last week of the treatment for 5 males and 5 (lactating) females/dose
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity (2 minutes) assessment and other functional 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 and ophthalmoscopy

IMMUNOLOGY: Yes
- Time schedule for examinations: in the last week of the treatment for all males and (lactating) females/dose
- Dose groups that were examined: males only
- Parameters checked: thyroxine (T4)

OTHER: estrous cycle
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes from 5 males and 5 females per dose groups (4 females at 600 mg/kg bw)
testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from all adult males and females (weighed after fixation (complete weight)), liver, kidneys (paired weight), spleen, adrenals (paired weight), brain, pituitary gland, heart
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table) A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period and the animal that died.

Other examinations:

estrous cycle:: monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Statistics:

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, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.1.3, Pathology Data Systems Ltd.).
A statistical assessment of the results of body weight, food consumption and litter data 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 1.1.3 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):

The main findings included diarrhoea, moving of bedding and increased salivation
Incidental findings are alopecia and abnormal breating.
The increased salivation may be related to the dosing procedures and thus dependent on dose applied

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 female at 600 mg/kg bw died (considered to a gavage error)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see table

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see table

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

checked under functional observations in the animals used for behavioural assessment (5/sex/dose) pre-test and during the week of termination. Data only available in individual tables

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males (n=5): statistically significantly higher WBC count in MD (an effect that was also seen in LD and HD) and significantly lower reticulocytes count in MD and HD. These effects were within historical control values (range WBC- 2.0- 8.2 109/L, reticulocytes- 1.1-2.4 %)
Females (n=4): statistically significantly lower group mean aPTT was observed in MD (can be attributed to a single female)
see table

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

males: no treatment related effects
females: non-significant increase of ASAT and ALAT (see table) that is not toxicologically relevant. (Historical control data range- Male- ALAT: 14.8- 126.5 U/L, ASAT: 42.1 -129.3 U/L; Female- ALAT: 4.6- 113.7 U/L, ASAT: 30.3 -148.1 U/L).

Urinalysis findings:

no effects observed

Description (incidence and severity):

only table with individual data available (see table)
Incidental high protein levels in all dose groups and controls

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
Only individual tables available for most parameters (see tables)

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There is a non-significant decrease in T4 concentratons in high dose males (12%). This decrease is within historical control values (19.2- 127 nmol/L).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increased abs and/or rel liver weights in males and females at mid dose and high dose
Decreased ovary weight at low dose females
(see table)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The female that died: fluid filled thoracic cavity, thymus abnormal colour (brown/orange)
One female in low dose group: thymus abnormal colour (brown/orange) and axillary lymph nodes abnormal colour (red)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The effects in the two females with macroscopic findings was attributed to congestion
Additional findings in the high dose animals included Stomach vacuolisation, Liver hepatocellular hypertrophy and Kidney hyaline inclusions (see table). These were considered not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pre-mating estrous cycle (at least mean length and frequency of irregular cycles) in days: 4.00, 4.10, 4.17 and 4.10 d for control low does, mid dose and high dose

Details on results:

During the treatment period of this study, one mortality was observed in high dose females. Histopathologically, the cause of death was not evident. In this animal, fluid filled thoracic cavity was observed at necropsy. Although there were no histological findings that could be related to the cause of death, it is considered that this animal died due to a technical gavage error and not due to systemic toxicity due to test item administration.
In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were sneezing in one animal of HD group on day 10 during mating/post mating period, diarrhoea in two each animals of control group (PMD 2-4) and MD group (PMD 3-4), slight to moderately increased salivation in one animal of LD (PMD 5-6), all animals of MD and HD during majority of days of premating, mating/postmating period and moving the bedding in 10 animals of HD group during mating and post mating day 4-12.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were slight to moderate increased salivation in two MD (few days between PMD 5- GD 17 in one and on PND 9 in second) and 9 HD group females (on majority of premating, gestation and post-natal days) and moving the bedding in one LD (on PND 9), 6 MD (occasionally during gestation and post-natal days) and in 9 HD females on majority of gestation and post-natal days.
There were also low incidences of the clinical signs like alopecia on various body parts of the 2 females of MD and 1 female of HD group, abnormal breathing in one HD female and diarrhoea in 2 HD females observed.
The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
None of the females showed signs of abortion or premature delivery.
During the weekly detailed clinical observation, no relevant differences between the groups were found.
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group.
In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.
Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.
No test item related effect of toxicological relevance or statistical significance was observed male thyroxine hormone (T4) in the treatment groups when compared to the controls or the historical data. T4 is decreased in both adult males (12% at HD). This effect cannot be attributed to an outlier. As the hormone underlies high interindividual variance and within historical control data range (19.2- 127 nmol/L), this is not considered to be test item related.
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group. However, statistically significantly higher WBC count in MD reaching significance and also in the LD and HD (but not reaching statistical significance) and lower reticulocytes count in MD and HD was observed when compared with the controls. As all values were within historical data range and there was no effect on RBC values, this effect on reticulocytes was not considered to be of toxicological relevance.
In females (only lactating females) sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters.
All other group mean and most of the individual values for haematological parameters in male and females were within the historical control data range.
No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls. However, statistically significantly lower group mean aPTT was observed in MD group females when compared with the controls. This value can be attributed to a single female (dam no. 69) and is not considered test item related.
In males and females (only lactating females) sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control.
All group mean and most of the individual values for clinical chemistry parameters in male and females were within the historical control data range. ALAT and ASAT are increased in the HD animals (although not statistically significant; mean increase is 12 and 14% resp. these effects cannot be attributed to an outlier). However, the increases in ASAT and ALAT are minute and not toxicologically relevant. Adversity is seen from 2- or 3-fold upwards.
A tendency towards a higher mean TBA level in male – but not female animals of the HD group is not considered toxicologically relevant. Due to high variability in this parameter this is assumed to be incidental.
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.
Few specific macroscopic changes were recorded in the female animals, which based on microscopic examination were not considered to be of test item treatment relevance. No macroscopic findings were recorded in any male animal.
The predominant macroscopic changes observed were fluid filled thoracic cavity (female no. 80 of HD group), thymus abnormal colour- brown/orange (female no. 54 of LD group and females no. 80 of HD group) and axillary lymph nodes- abnormal colour red (female no. 54 of LD group).
In animal no. 54, the abnormal colour (brown/red) thymus and axillary lymph nodes and in animal no. 80, abnormal colour (orange) thymus histologically correlated with congestion.
The above mentioned findings were deemed incidental and there were no gross lesions that could be attributed to treatment with the test item.
In males statistically significantly higher absolute liver weights in HD group and relative liver weights in MD and HD were observed when compared with the controls. In the light of fact that no test item related histopathological findings and effects on liver enzymes were observed, this increase in male liver weight was not considered to be adverse.
In females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group except statistically significantly higher absolute and relative ovary weights were observed in LD group females when compared with the controls. In the light of fact that no test item related histopathological findings and due to lack of dose dependency, this increase in female ovary weight was not considered to be test item related.
Test item did not produce morphological or histological evidence of toxicities in the organs and tissues examined in this study. Single findings as stomach vacuolization, liver with inflammatory or hemopeoietic foci, or hyaline inclusions in the kidneys were also seen in the control animals and are not considered to be test item related.
The test item did not produce histological evidence of toxicity in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, and vagina.
The sperm staging did not reveal any treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. Sperm staging revealed sperm re-absorption in one single stage V tubule in male no. 2. Re-absorption was also observed in one single stage VIII tubule in animal nos. 4, 6, 9, 33, and 39. These findings are of no pathological consequence but are within the range of normal changes that may be observed in animals at these ages. There were not any abnormality in any testis examined. There were no indications for sperm arrest, incomplete maturation, reabsorption etc. All sperm stages were complete.
There were no non-pregnant animals in intermediate groups. In conclusion, a histopathological NOEL (No Observed Effect Level) could be established at 600 mg//kg bw/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects observed

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Dose	0		100		300		600		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Mortality	0/10	0/10	0/10	0/10	0/10	0/10	0/10	1/10	No
Clinical signs -diarrhoea -moving of bedding -increased salivation	2 (pre-mat)			1 (lact)	2 (pre-mating)   10 ((post)mating)	6 (gest/lact) 2 (pre-mating /gest/lact)	9 ((post)mating) 10 ((post)mating)	2 (gest/lact) 9 (gest/lact) 10 (pre-mating /gest/lact)	Yes (salivation)
Body weight (gain)	NTRE								No
Food consumption	NTRE								No
Behavioral effects (pre-test +post-mating/lact)	NTRE								No
Motoractivity (during 2 min)	NTRE								No
Estrous cycle (pre-test)	NTRE (4 days)								No
Pre-coital interval	NTRE (1-4 days)								No
Thyroxine (T4) (males)							↓ (12% ns)		No (within hist control)
Conception rate (%)		100		100		100		88.9	No
Fertility index (%)		100		100		100		88.9	No
Gestation length	NTRE (21-23 days)								No
Gestation index (%)		100		100		100		100	No
Live litters		10		10		10		8	No
Implementation sites (mean)		11.4		11.2		12.6		11.3	No
Litter size (mean)		11.1		11.3#		12.0		11.1	No
Haematology			WBC↑(15% ns)		Retic↓ (19%) WBC ↑ (41%)	aPPT↓ (32%)	Retic↓ (23%)* WBC ↑ (20% ns)*		Within historical controls
Clinical biochemistry								ALAT↑ (12% ns)* ASAT ↑ (14% ns)*	No
Organ weights				Ovaries (rel) ↑ (22%)	liver (rel)↑ (17% )	liver (rel) ↑ (15% ns)	liver↑ (30%) prostate ↓ (13% ns)	liver (rel)↑ (11% ns)	No (incidental)
Marcoscopy	NTRE								No
Histopathology Stomach vacuolisation Liver hepatocellular hypertrophy Kidney hyaline inclusions	0/5 0/5 4/5	0/5 0/5 0/5					5/5 4/5 5/5	0/5 3/5 0/5	No

NTRE= no treatment related effects

# measured in 9 litters versus 10 for number of implantation sites

↑/↓= significantly increased/decreased at 1% or 5% level

*based on 5 males and 4 females

% compared to controls

Body weight

		Males		Females
				Premating		Gestation		Lactation
Group		Day 1	Terminal Sacrifice	Day 1	Day 14	Day 0	Day 20	Day 0	Day 13
C	Mean	423.70	450.60	245.50	255.30	254.67	361.70	290.80	309.20
	SD	36.14	43.73	10.79	13.19	14.12	19.64	19.50	17.57
	N	10	10	10	10	9	10	10	10
LD	Mean	421.70	451.10	243.20	253.70	254.50	364.10	292.75	305.00
	SD	38.42	37.34	13.02	16.77	16.23	29.47	22.58	20.62
	N	10	10	10	10	10	10	8	10
	%	100	100	99.1	99.4	99.9	100.7	100.7	98.6
MD	Mean	422.70	458.50	250.70	259.20	256.20	374.90	300.56	322.70
	SD	42.53	44.62	15.12	14.85	15.92	18.15	19.90	15.08
	N	10	10	10	10	10	10	9	10
	%	100	102	102.1	101.5	100.6	103.6	103.4	104.4
HD	Mean	422.30	442.80	246.90	254.11	251.63	364.13	291.75	311.25
	SD	33.18	37.05	12.67	13.20	16.26	16.11	23.87	24.03
	N	10	10	10	9	8	8	8	8
	%	100	98	100.6	99.5	98.8	100.7	100.3	100.7

 

Food consumption

		Males		Females
				Premating		Gestation			Lactation
Group		Day 1-7	Day 7-14	Day 1-7	Day 7-14	Day 0-7	Day 7-14	Day 14-20	Day 0-4	Day 4-9	Day 4-13
C	Mean	135.50	172.90	93.80	113.70	128.11	138.90	134.10	118.20	225.00	196.20
	SD	8.63	1.84	1.98	1.27	11.27	14.86	17.54	18.43	37.40	20.43
	N	2	2	2	2	9	10	10	10	10	10
LD	Mean	132.40	161.20	95.50	112.10	119.90	141.50	139.00	113.50	217.20	188.10
	SD	5.66	4.81	3.54	7.78	10.73	12.80	21.41	17.60	21.57	20.75
	N	2	2	2	2	10	10	10	8	10	10
MD	Mean	134.70	163.60	93.20	112.90	120.30	144.90	137.30	123.44	252.00	218.90
	SD	4.95	5.94	3.39	7.78	9.04	12.58	6.31	8.47	17.80	19.19
	N	2.00	2.00	2	2	10	10	10	9	10	10
HD	Mean	127.80	164.90	102.50	112.23	114.00	132.63	132.63	113.86	227.86	193.71
	SD	13.01	0.14	10.32	1.45	15.26	18.20	9.16	20.12	37.75	27.71
	N	2	2	2	2	8	8	8	7	7	7

 

 

 

Urinalysis males

Group

Animal No.

Ery

UBG

BIL

Protein

Nitrite

Ket

Glucose

pH

Specific Gravity

Leuc

Colour / Appearance

Units

cells/µL

mg/dL

mg/dL

mg/dL

-

mg/dL

mg/dL

-

-

cells/µL

-

C

3

neg.

norm.

neg.

neg.

neg.

neg.

20

8

1.005

neg.

colourless

4

neg.

norm.

neg.

neg.

neg.

neg.

20

8

1.005

neg.

yellowish

5

ca.250

norm.

neg.

neg.

neg.

neg.

20

8

1.005

ca.25

colourless

7

neg.

norm.

neg.

30

neg.

25

neg.

8

1.005

ca. 25

yellowish

9

ca. 10

norm.

neg.

30

neg.

neg.

neg.

8

1.000

neg.

colourless

LD

12

ca.10

norm.

neg.

100

neg.

25

neg.

9

1.000

ca.25

colourless

14

neg.

norm.

neg.

30

neg.

25

neg.

8

1.005

ca.25

colourless

15

ca.10

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

colourless

16

ca.50

norm.

neg.

100

neg.

25

neg.

8

1.015

ca.500

yellowish

18

ca.10

norm.

neg.

30

neg.

neg.

neg.

9

1.000

ca.25

colourless

MD

22

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

yellowish

26

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

yellow

27

no urine available

28

no urine available

29

neg.

norm.

neg.

100

neg.

25

neg.

8

1.005

ca.25

yellowish

HD

31

neg.

norm.

neg.

neg.

neg.

25

neg.

8

1.005

ca.25

yellowish

33

ca.50

norm.

neg.

30

neg.

25

20

8

1.005

ca.25

yellowish

34

neg.

norm.

neg.

30

neg.

25

neg.

8

1.005

neg.

yellow

37

neg.

norm.

neg.

neg.

neg.

neg.

neg.

8

1.000

neg.

yellowish

38

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

yellowish

Urinalysis Females

Group

Animal No.

Ery

UBG

BIL

Protein

Nitrite

Ket

Glucose

pH

Specific Gravity

Leuc

Colour / Appearance

Units

cells/µL

mg/dL

mg/dL

mg/dL

-

mg/dL

mg/dL

-

-

cells/µL

-

C

41

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

43

neg.

norm.

neg.

neg.

neg.

neg.

neg.

8

1.005

ca.25

yellowish

44

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

yellowish

45

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

yellowish

48

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

LD

51

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

53

neg.

norm.

neg.

30

neg.

25

neg.

8

1.015

ca.25

yellowish

54

neg.

norm.

neg.

30

neg.

25

neg.

8

1.005

neg.

yellow

55

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

59

neg.

norm.

1

100

neg.

25

neg.

8

1.005

ca.25

yellow

MD

61

neg.

norm.

neg.

neg.

neg.

neg.

neg.

8

1.005

neg.

colourless

62

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

68

neg.

norm.

1

100

neg.

25

neg.

8

1.005

25

yellowish

69

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

yellowish

70

ca.250

norm.

neg.

30

neg.

neg.

neg.

9

1.000

ca.25

colourless

HD

72

neg.

norm.

neg.

100

neg.

neg.

neg.

8

1.005

neg.

yellowish

77

neg.

norm.

neg.

neg.

neg.

neg.

neg.

7

1.005

neg.

colourless

78

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

79

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

colourless

-

-

-

-

-

-

-

-

-

-

-

-

neg. = negative; pos. = positive; norm. = normal; n.a. = no data available

Functional Observations – Males – Summary

Group		Body Temperature (°C)		Rearing Supported (Count)		Rearing not Supported (Count)		Urination (Count)		Defecation (Count)
		Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment
C	Mean	38.16	38.10	5.20	2.00	1.40	0.20	3.20	2.20	2.60	3.00
	SD	0.21	0.27	3.35	2.83	1.34	0.45	1.79	1.48	1.67	2.74
	N	5	5	5	5	5	5	5	5	5	5
LD	Mean	38.32	37.62	4.00	2.60	0.60	0.20	3.60	0.60	2.40	1.20
	SD	0.30	0.23	2.55	0.89	0.55	0.45	2.51	0.55	2.07	1.64
	N	5	5	5	5	5	5	5	5	5	5
MD	Mean	38.06	37.72	4.00	3.20	0.60	0.40	3.60	1.60	2.00	2.00
	SD	0.09	0.53	2.12	1.10	0.89	0.89	2.88	1.14	1.87	1.22
	N	5	5	5	5	5	5	5	5	5	5
HD	Mean	38.28	37.44	3.80	2.00	1.40	0.20	3.20	1.00	3.20	2.00
	SD	0.26	0.94	1.48	1.87	2.07	0.45	3.11	1.00	1.10	1.00
	N	5	5	5	5	5	5	5	5	5	5

Functional Observations – Females – Summary

Group		Body Temperature (°C)		Rearing Supported (Count)		Rearing not Supported (Count)		Urination (Count)		Defecation (Count)
		Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment
C	Mean	38.18	38.28	4.00	5.40	1.00	0.80	2.60	0.80	4.20	1.40
	SD	0.19	0.22	1.58	0.89	1.22	0.84	2.41	0.84	1.30	1.34
	N	5	5	5	5	5	5	5	5	5	5
LD	Mean	38.18	38.36	5.80	5.60	1.20	0.40	5.20	0.60	2.00	1.00
	SD	0.31	0.11	1.10	1.14	1.30	0.89	2.17	0.89	1.87	1.00
	N	5	5	5	5	5	5	5	5	5	5
MD	Mean	38.16	38.46	5.80	5.80	1.20	0.60	2.60	0.80	4.40	1.40
	SD	0.50	0.11	1.64	1.48	1.79	1.34	2.61	1.30	2.70	1.34
	N	5	5	5	5	5	5	5	5	5	5
HD	Mean	37.92	38.40	5.00	5.25	1.00	0.75	3.40	1.25	3.00	1.00
	SD	0.23	0.18	1.58	1.71	0.71	0.96	3.58	1.89	2.65	1.15
	N	5	4	5	4	5	4	5	4	5	4

 

T4 analyses Males

Group		Male- Thyroxine (T4)
	Units	nmoL/L
C	Mean	83.35
	SD	10.84
	N	10
LD	Mean	76.62
	SD	4.41
	N	10
MD	Mean	77.66
	SD	7.52
	N	10
HD	Mean	73.82
	SD	15.45
	N	10

 

 

Applicant's summary and conclusion

Conclusions:

Based on the absence of treatment related adverse effects the NOAEL is 600 mg/kg bw.

Executive summary:

The test item was administered daily at 0, 100, 300 and 600 mg/kg bw (in PEG400) during 14 days of pre-mating 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 until the minimum total dosing period of 28 days were completed.

During the period of administration, the animals were observed each day for signs of toxicity, body weight and food consumption were measured. Functional observations were performed for all animals before treatment and in five males and females in the last week of treatment

Haematological, clinical biochemistry and urine investigation were performed on selected males and females from each group. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days.

The males were sacrificed after completion of the mating period on treatment day 29 and the females were sacrificed on post natal day 13.

A full histopathological evaluation of the preserved tissues was performed on high dose and control animals and dead animal. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in any organ/tissues of the high dose group. 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.All gross lesions macroscopically identified were examined microscopically in all animals.

During the treatment period of this study, one mortality was observed in high dose females.Histopathologically,the cause of death was not evident.In this animal, fluid filled thoracic cavity was observed at necropsy. Although there were no histological findings that could be related to the cause of death, it is considered that this animal died due to a technical gavage error and not due to systemic toxicity due to test item administration.

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were sneezing in one animal of HD group on day 10 during mating/post mating period, diarrhoea in two each animals of control group (PMD 2-4) and MD group (PMD 3-4), slight to moderately increased salivation in one animal of LD (PMD 5-6), all animals of MD and HD during majority of days of premating, mating/postmating period and moving the bedding in 10 animals of HD group during mating and post mating day 4-12.

In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were slight to moderate increased salivation in two MD (few days between PMD 5- GD 17 in one and on PND 9 in second) and 9 HD group females (on majority of premating, gestation and post-natal days) and moving the bedding in one LD (on PND 9), 6 MD (occasionally during gestation and post-natal days) and in 9 HD females on majority of gestation and post-natal days.

There were also low incidences of the clinical signs like alopecia on various body parts of the 2 females of MD and 1 female of HD group, abnormal breathing in one HD female and diarrhoea in 2 HD females observed.

The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group.

In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group.No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

No test item related effect of toxicological relevance or statistical significance was observed male thyroxine hormone (T4) in the treatment groups when compared to the controls or the historical data. T4 is decreased in both adult males (12% at HD). This effect cannot be attributed to an outlier. As the hormone underlies high interindividual variance and within historical control data range (19.2- 127 nmol/L), this is not considered to be test item related.

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group. However, statistically significantly higher WBC count in MD reaching significance and also in the LD and HD (but not reaching statistical significance) and lower reticulocytes count in MD and HD was observed when compared with the controls. As all values were within historical data range and there was no effect on RBC values, this effect on reticulocytes was not considered to be of toxicological relevance.

In females (only lactating females) sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology parameters.

All other group mean and most of the individual values for haematological parameters in male and females were within the historical control data range.

No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls. However, statistically significantly lower group mean aPTT was observed in MD group females when compared with the controls. This value can be attributed to a single female (dam no. 69) and is not considered test item related.

In males and females(only lactating females)sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control.

All group mean and most of the individual values for clinical chemistry parameters in male and females were within the historical control data range.ALAT and ASAT are increased in the HD animals (although not statistically significant; mean increase is 12 and 14% resp. these effects cannot be attributed to an outlier).However, the increases in ASAT and ALAT are minute and not toxicologically relevant. Adversity is seen from 2- or 3-fold upwards.

A tendency towards a higher mean TBA level in male – but not female animals of the HD group is not considered toxicologically relevant. Due to high variability in this parameter this is assumed to be incidental.

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Few specific macroscopic changes were recorded in the female animals, which based on microscopic examination were not considered to be of test item treatment relevance.No macroscopic findings were recorded in any male animal.

The predominant macroscopic changes observed were fluid filled thoracic cavity (female no. 80 of HD group), thymus abnormal colour- brown/orange (female no. 54 of LD group and females no. 80 of HD group) and axillary lymph nodes- abnormal colour red (female no. 54 of LD group). The above mentioned findings were deemed incidental and there were no gross lesions that could be attributed to treatment with the test item.

In males statistically significantly higher absolute liver weights in HD group and relative liver weights in MD and HD were observed when compared with the controls. In the light of fact that no test item related histopathological findings and effects on liver enzymes were observed, this increase in male liver weight was not considered to be adverse.

In females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group except statistically significantly higher absolute and relative ovary weights were observed in LD group females when compared with the controls. In the light of fact that no test item related histopathological findings and due to lack of dose dependency, this increase in female ovary weight was not considered to be test item related.

Test item did not produce morphological or histological evidence of toxicities in the organs and tissues examined in this study. Single findings as stomach vacuolization, liver with inflammatory or hemopeoietic foci, or hyaline inclusions in the kidneys were also seen in the control animals and are not considered to be test item related.

The test item did not produce histological evidence of toxicity in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus, cervix, and vagina.

The sperm staging did not reveal any treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.There were not any abnormality in any testis examined. There were no indications for sperm arrest, incomplete maturation, reabsorption etc. All sperm stages were complete.

There were no non-pregnant animals in intermediate groups.

In conclusion in absence of treatment related adverse effects the NOAEL is set at 600 mg/kg bw.