Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
repeated dose reproduction study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September 2017 to 11 April 2018 ??????
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Develop mental Toxicity Screening Test. EPA 712-C-00-368, July 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate
EC Number:
281-468-5
EC Name:
4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate
Cas Number:
83950-14-5
Molecular formula:
C24H27N2O.C2H3O2
IUPAC Name:
4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate
Test material form:
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: 344-420 g; females: 222 - 258 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 2
- 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:
water
Remarks:
water ad injectionem
Details on 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. 173717). The prepared formulation was stored at room temperature. Formulates were kept under magnetic stirring during the daily administration.

- VEHICLE: water (aqua ad injectionem)
-Dosing volume: 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal smear that is sperm-positive, day with vaginal plug and/or sperm was considered as day 0 of gestation.
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no
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: 30°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
0.01 85 15
8.00 5 95
10.00 5 95
12.00 85 15
14.00 85 15
Run time: 14 min
Retention time: Approx. 5.4 min, main component
Detection: 254 nm

Calibration: 0.02-0.52 mg/mL linear r= 0.99996 (accuracy 99.1-101.1% of spiked)
QC samples (11.2, 21.4 and 50.3 mg/mL): 97.5-100.8% of nominal (COV 0.40-2.47%)
Stability (11.2 and 50.3 mg/mL):: 90.3-100.2% and 92.8-99.2% (stability measured over 6 hours, RT; 12 days, RT; 12 days, 2-8°C ;12 days, -15 to -35°C)
Homogeneity (11.2 and 50.3 mg/mL mg/mL): 100% (COV 0.7%) and 97.5% (COV 0.2%)

Accuracy of concentrations applied (see attached document)
control: NA
10 mg/mL: 100.5 ± 0.4%
20 mg/mL: 100.2 ± 0.9%
50 mg/mL: 100.9 ± 0.8%
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 postmating
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale: based on a dose range finding study (see below)
On the basis of this dose range finding reproduction/ developmental toxicity screening test with C.I. Basic Orange 60 in male and female Wistar rats (3/sex/dose) with dose levels of 100, 300 and 800 mg/kg body weight day the following conclusions can be made:
Due to worsened health and mortality both the mid dose and the high dose were lowered for survivors on day 8 to 200 mg/kg bw.
After this reduction there were slight effects on body weight and food consumption in females during lactation treated at 200 mg/kg bw. At 300/200 and 800/200 mg/kg bw orange discoloration of feaces was reported from day 6 throughout the study period. There were no adverse clinical symptoms observed during the treatment period. At necropsy, there were no macroscopic findings and no effects on organ weights.
There was a slightly lower number of pups and implantation sites and increased preimplantation loss in 1/3 females at 200 mg/kg bw. Other post-natal parameters including the number of corpora lutea were not affected. There were no other litter parameters including the sex ratio, total litter weight, male litter and female litter weight affected.
Based on the generated data the dose levels 50, 100 and 250 mg/kg bw were considered for the main OECD 422 study with C.I. Basic Orange 60.
Positive control:
NA

Examinations

Parental animals: Observations and examinations:
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 5 (lactating) females/dose from abdominal aorta (control 4 females, 50 mg/kg bw 3 females)
- Anaesthetic used for blood collection: ketamine/xylazine
- 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 5 (lactating) females/dose from abdominal aorta (50 mg/kg bw 4 females)
- Anaesthetic used for blood collection: ketamine/xylazine
- 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 assessments 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: Precoital Interval and Duration of Gestation
Oestrous cyclicity (parental animals):
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.
In addition vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, testes tubular stages of the spermatogenic cycle (PAS (Periodic Acid Schiff) stained slides)
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, litter weight (weight gain) on day 0, 4 and 13, physical or behavioural abnormalities, anogenital distance (AGD) on day 0, presence of nipples/areolae in male pups on day 12:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:measurement of thyroxine (T4) in 2 pups/litter at termination on day 13
Weight of thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13)
Postmortem examinations (parental animals):
ORGAN WEIGHTS: Yes from 5 males and 5 females per dose groups
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.
Ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.

REPRODUCTIVE PARAMETERS: Yes
Precoital interval, gestation length, corpora lutea, implantation sites
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS AND SURVIVING PUPS:
yes, for external abnormalities
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).
Reproductive indices:
Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100;
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100;
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100;
Offspring viability indices:
Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The main findings included diarrhoea, discoloured faeces, moving of bedding and increased salivation that increased in incidence with dose
see table repeated dose toxicity
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Four female animals died during the treatment period. Animal no. 52 in the LD group was found dead on premating day 5, animal no. 62 in the MD group on mating day 1 and in the HD group animal no. 71 on premating day 2 and animal no. 80 on premating day 10. All remaining animals survived their scheduled study period.
Histopathologically, there was no morphological indicator for toxicity in any organ or tissue examined. However, due to the detection of alveolar fluid in the lungs of one descendent animal at histopathological evaluation (animal no. 80) and findings seen at necropsy (animal no. 52 and 62), it is considered that gavage error might have caused death in these female animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no effects observed (see table repeated dose study)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no effects observed (see table repeated dose toxicity)
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: at 250 mg/kg bw non-sign decreased aPTT (-32%)
Females: no treatment related effects

see table repeated dose toxicity
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
males: non significant effects on ALAT, ASAT, ALP, urea and bile acids (see table repeated dose toxicity)
females: at 50 mg/kg bw ASAT sign decrease (32%); at 250 mg/kg bw ASAT sign decrease (29%) within historical control values and without dose effect relationship; non-significant effects on ALAT and bile acids (see table repeated dose toxicity)

(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
High protein levels in male and females of all groups including control group.
Slightly elevated levels of bilirubin in one male animal of the HD group and urobilinogen in one female HD animal are considered to be without toxicological relevance as there were no correlating clinical chemical or histopathological findings observed
Behaviour (functional findings):
no effects observed
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 table repeated dose toxicity)
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no histological findings that distinguished controls from test item-treated animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrus cycle in days: 4.00, 4.00, 4.00 and 4.17 d for control low does, mid dose and high dose.
A cycle length of more than 5 days observed for one animal in the HD group (animal no. 79) is considered to be incidental.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no change at all noted during sperm staging. The stages were complete and avoid of any degenerative change.

Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Copulation Index (%) 100, 100, 100, 100 for controls, low dose, mid dose and high dose animals
Fertility Index (%): 100, 100, 89 and 100 for controls, low dose, mid dose and high dose animals (1 female at the mid dose group was not pregnant)
Delivery Index (%): 100, 100, 100, 100 for controls, low dose, mid dose and high dose animals

Precoital interval (mean) 2.00, 2.10, 1.70 and 2.33 days for controls, low dose, mid dose and high dose animals

Gestation length (mean) 22.40, 22.67, 22.13 and 22.50 days or controls, low dose, mid dose and high dose animals

Corpora lutea/dam (mean) 13.4, 13.7, 12.9 and 13.5 for controls, low dose, mid dose and high dose animals

Implants/dam (mean) 13.20, 13.56, 12.88 and 13.25 for controls, low dose, mid dose and high dose animals

Pre-implementation loss (%) 1.43, 0.85, 0.0 and 1,67 for controls, low dose, mid dose and high dose animals

Post implementation loss (%) 7.78, 14.2, 3.85 and 8.25 for controls, low dose, mid dose and high dose animals

Details on results (P0)

No mortality occurred in the male control or any of the male dose groups during the treatment period of the study. Four female animals died during the treatment period most likely due to gavage errors. Clinical signs that could be related to treatment include increased salivation, discoloration of the faec
es, diarrhea and abnormal breathing. 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. In addition there were no effects on body weight and food consumption. Effects on blood were limited to a decreased aPTT in high dosed males. Clinical biochemistry findings included a significant decrease of ASAT in females at 50 and 250 mg/kg bw , that was within historical control data. Parameters measured during urine analysis were within normal ranges. In survivors there were no treatment related macroscopic effects. Liver weights in females at 100 and 250 mg/kg bw were significantly increased but there were no histopathological findings in these livers. In absence of a clear dose response this effect was considered not related to treatment. Other effects on organ weights were considered incidental and not related to treatment. 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
There were no test item treatment related effects observed on gestation length, the number of corpora lutea, implantation sites, live pups born, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.
There were no test item related effects on the reproductive indices (copulation, fertility and delivery indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 88.89 % was observed in the MD group as compared to 100 % in control group. Although there was reduction in fertility index in MD group, it was within the standard pregnancy rate of rat i.e. ≥ 80 % and therefore this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
250 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 (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
see table
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see table
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
thyroids of 2 pups per litter were weighed (see table)
Gross pathological findings:
no effects observed
Description (incidence and severity):
No substance related gross external abnormalities of toxicological relevance on PND 0-12 and at death were observed in the pups of any of the groups. Few specific findings like dark tail tip (pup no. 4 from dam 57 of the LD group from PND 8-12 until death), alopecia on back and head (all pups of animal no. 44 and 46 of the C group at death), one lean pup of animal no. 57 in the LD group and alopecia on the back of all pups of animal no. 69 in the HD group at death were observed and considered to be spontaneous when seen at death. One male pup (pup no. 12 from dam no. 60 of the LD group) was found dead on PND 0, which is considered to be an incidental finding.


Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance: no effects (see table)
Nipple count: Mean values of pup nipple retention in males on PND 12 were slightly to moderately increased in the dose groups (LD: 0.43, MD: 0.48, HD: 0.68) when compared to control (C: 0.35), but no statistical significance was achieved. Therefore a test item related effect on nipple retention is not considered.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
No effect on thyroid weight in 2 pups/litter
T4 in pups at day 13 (see table): decreased at high dose (20%). This decrease is within historical control values (19.2- 127 nmol/L).

Details on results (F1)

No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group.
In males and females, no statistically significant dose related effect on pup weight, cube root of pup weight on the day of anogenital measurement, absolute and relative anogenital distance in treatment groups when compared to the controls.
No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.
No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls or the historical data.
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Study Summary

OBSERVATIONS

 

Values

Groups

 

C

LD

MD

HD

Pairs started (N)

 

10

9

10

8

Oestrus cycle (at least mean length and frequency
of irregular cycles)

 

4.00

4.00

4.00

4.17

Females showing evidence of copulation (N)

 

10

9*

9**

8***

Females achieving pregnancy (N)

 

10

9

8

8

Conceiving days 1 - 5 (N)

 

10

9

8

7

Conceiving days 6 - . . .(1) (N) and more

 

0

0

1

1

Pregnancy≤21 days (N)

 

0

0

0

0

Pregnancy = 22 days (N)

 

6

3

7

4

Pregnancy≥23 days (N)

 

4

6

1

4

Dams with live young born (N)

 

10

9

8

8

Dams with live young at day 4 pp (N)

 

10

9

8

8

Corpora lutea/dam (mean)

 

13.40

13.67

12.88

13.50

Implants/dam (mean)

 

13.20

13.56

12.88

13.25

Live pups/dam at birth (mean)

 

12.20

11.56

12.38

12.13

Live pups/dam at day 4 (mean)

 

12.20

11.56

12.38

12.13

Sex ratio (m/f) at birth (mean)

 

0.92

1.15

1.31

1.01

Sex ratio (m/f) at day 4 (mean)

 

0.92

1.12

1.31

1.01

Pup weight at birth (mean)

 

6.36

6.78

6.11

6.28

Pup weight at the time of AGD measurement - PND 0
(mean males, mean females)

Male

6.65

6.96

6.29

6.39

Female

6.11

6.49

5.87

6.14

Pup AGD on PND 0 (mean males, mean females)

Male

2.57

2.65

2.57

2.52

Female

0.63

0.66

0.66

0.67

Pup weight at day 4 (mean)

 

11.20

11.86

10.79

11.02

Male pup nipple retention at day 12 (mean)

 

0.35

0.43

0.48

0.68

Pup weight at day 13 (mean)

 

28.95

30.25

28.19

29.05

Litter weight at birth (mean)

 

77.50

77.62

75.51

75.94

Litter weight at day 4 (mean)

 

135.92

135.16

133.18

132.71

ABNORMAL PUPS

 

 

 

 

 

Dams with 0

 

10

7

8

8

Dams with 1

 

0

2

0

0

Dams with 2 and more

 

0

0

0

0

LOSS OF OFFSPRING

 

 

 

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

 

 

 

Females with 0

 

9

8

8

6

Females with 1

 

0

1

0

2

Females with 2

 

1

0

0

0

Females with 3

 

0

0

0

0

Pre-natal (implantations minus live births)

 

 

 

 

 

Females with 0

 

4

1

5

2

Females with 1

 

4

4

2

4

Females with 2

 

0

2

1

1

Females with 3 and more

 

2

2

0

1

Post-natal (live births minus alive at post natal day 4)

 

 

 

 

 

Females with 0

 

10

9

8

8

Females with 1

 

0

0

0

0

Females with 2

 

0

0

0

0

Females with 3 and more

 

0

0

0

0

Post-natal (alive at post natal day 4 minus alive at post natal day 13)

 

 

 

 

 

Females with 0

 

10

9

8

8

Females with 1

 

0

0

0

0

Females with 2

 

0

0

0

0

Females with 3 and more

 

0

0

0

0

(1) = last day of mating period; *= one female (no. 52) dead on premating day 5;
**= one female (no. 62) dead on mating day 1;
***- one female (no. 71) dead on premating day 2, one female (no. 80) dead on premating day 10


 

 

 

 

 

Pre- and Post-Natal Data

Group

 

Corpora Lutea (CL)

Implantation Sites (IS)

Live Pups on PND 0

Live Pups on PND 4 (before)

Live Pups on PND 4 (after)

Live Pups on PND 13

Pre Implantation Loss (%)

Post Implantation Loss (%)

C

Mean

13.40

13.20

12.20

12.20

10.30

10.30

1.43

7.78

SD

1.43

1.48

1.99

1.99

1.83

1.83

4.52

8.76

N

10

10

10

10

10

10

10

10

LD

Mean

13.67

13.56

11.56

11.56

9.78

9.78

0.85

14.12

SD

1.41

1.51

1.81

1.81

1.39

1.39

2.56

13.69

N

9

9

9

9

9

9

9

9

MD

Mean

12.88

12.88

12.38

12.38

10.38

10.38

0.00

3.85

SD

1.13

1.13

1.30

1.30

1.30

1.30

0.00

5.81

N

8

8

8

8

8

8

8

8

HD

Mean

13.50

13.25

12.13

12.13

10.25

10.25

1.67

8.28

SD

1.77

1.58

1.46

1.46

1.16

1.16

3.09

6.72

N

8

8

8

8

8

8

8

8

 

Mean Pup Weight

Group

 

Pup Mean weight on Day 0 (g)

Pup Mean weight on Day 4 (g)

Pup Mean weight on Day 13 (g)

C

Mean

6.36

11.20

28.95

SD

0.43

0.89

1.97

N

10

10

10

LD

Mean

6.78

11.86

30.25

SD

0.65

1.53

3.74

N

9

9

9

MD

Mean

6.11

10.79

28.19

SD

0.27

0.61

1.31

N

8

8

8

HD

Mean

6.28

11.02

29.05

SD

0.42

0.93

2.31

N

8

8

8

Mean litter weight

 

PND 0

PND 4

PND 13

Group

 

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

C

Mean

77.50

36.55

40.95

135.92

63.69

72.23

296.33

160.53

135.80

SD

13.35

16.05

10.66

20.86

27.71

18.03

42.66

64.10

51.56

N

10

10

10

10

10

10

10

10

10

LD

Mean

77.62

39.42

38.20

135.16

68.17

66.99

292.06

171.87

120.19

SD

9.01

11.64

11.00

15.26

19.78

18.08

25.77

49.51

44.46

N

9

9

9

9

9

9

9

9

9

MD

Mean

75.51

42.49

33.03

133.18

74.79

58.39

291.81

192.75

99.06

SD

7.10

5.02

8.33

12.00

11.28

13.47

31.09

26.27

35.82

N

8

8

8

8

8

8

8

8

8

HD

Mean

75.94

38.34

37.60

132.71

66.21

66.49

296.05

173.46

122.59

SD

8.39

7.81

8.29

11.88

12.62

12.88

23.64

32.45

32.21

N

8

8

8

8

8

8

8

8

8

 

 

Mean Pup Survival Data (day 1-4)

Group

 

Post Natal Day

Total Mortality (PND 0-4) %

1

4

Number of alive Males

Number of alive Females

Total

Number of alive Males

Number of alive Females

Total

C

Mean

5.50

6.70

12.20

5.50

6.70

12.20

0.00

SD

2.22

2.00

1.99

2.22

2.00

1.99

0.00

N

10

10

10

10

10

10

10

LD

Mean

5.67

5.89

11.56

5.67

5.89

11.56

0.00

SD

1.58

2.15

1.81

1.58

2.15

1.81

0.00

N

9

9

9

9

9

9

9

MD

Mean

6.75

5.63

12.38

6.75

5.63

12.38

0.00

SD

0.71

1.41

1.30

0.71

1.41

1.30

0.00

N

8

8

8

8

8

8

8

HD

Mean

6.00

6.13

12.13

6.00

6.13

12.13

0.00

SD

1.41

0.99

1.46

1.41

0.99

1.46

0.00

N

8

8

8

8

8

8

8

 

Mean Pup Survival Data (day 4-13)

Group

 

Post Natal Day

Total Mortality (PND 4-13) %

4

13

Number of alive Males after interim sacrifice

Number of alive Females after interim sacrifice

Total

Number of alive Males

Number of alive Females

Total

C

Mean

5.50

4.80

10.30

5.50

4.80

10.30

0.00

SD

2.22

1.99

1.83

2.22

1.99

1.83

0.00

N

10

10

10

10

10

10

10

LD

Mean

5.67

4.11

9.78

5.67

4.11

9.78

0.00

SD

1.58

1.90

1.39

1.58

1.90

1.39

0.00

N

9

9

9

9

9

9

9

MD

Mean

6.75

3.63

10.38

6.75

3.63

10.38

0.00

SD

0.71

1.41

1.30

0.71

1.41

1.30

0.00

N

8

8

8

8

8

8

8

HD

Mean

6.00

4.25

10.25

6.00

4.25

10.25

0.00

SD

1.41

0.89

1.16

1.41

0.89

1.16

0.00

N

8

8

8

8

8

8

8

 

Mean Anogenital Distance and Nipple Retention

Male Pups

Female Pups

Group

 

Pup Weight (g)

Cube Root of Pup Weight

Anogenital Distance (mm) of Pups

Relative Anogenital Distance Pups

Pup nipple retention (N) on PND 12

Pup Weight (g)

Cube Root of Pup Weight

Anogenital Distance (mm) of Pups

Relative Anogenital Distance Pups

C

Mean

6.65

1.88

2.57

1.37

0.35

6.11

1.83

0.63

0.35

SD

0.50

0.05

0.22

0.11

0.91

0.59

0.06

0.08

0.04

N

55

55

55

55

55

67

67

67

67

LD

Mean

6.96*

1.91*

2.65

1.39

0.43

6.49**

1.86**

0.66

0.35

SD

0.69

0.06

0.23

0.11

0.76

0.67

0.06

0.09

0.04

N

51

51

51

51

51

53

53

53

53

MD

Mean

6.29**

1.85**

2.57

1.39

0.48

5.87

1.80

0.66

0.36

SD

0.46

0.05

0.21

0.11

0.75

0.37

0.04

0.05

0.03

N

54

54

54

54

54

45

45

45

45

HD

Mean

6.39*

1.85*

2.52

1.36

0.68

6.14

1.83

0.67

0.36

SD

0.50

0.05

0.31

0.15

1.18

0.57

0.06

0.10

0.05

N

48

48

48

48

47

49

49

49

49

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

 

Mean Thyroxine (T4) Analysis –Pups (Day 13)

Group

 

Pup-Thyroxine (T4)

Units

nmoL/L

C

Mean

108.10

SD

26.53

N

10

LD

Mean

107.35

SD

27.42

N

10

MD

Mean

109.85

SD

24.74

N

8

HD

Mean

86.98

SD

9.35

N

8

 

Mean Organ Weight Thyroid Gland (Post Fixation) Pups - Day 13

Group

 

Male Pups - Thyroid/Parathyroid glands

Female Pups - Thyroid/Parathyroid glands

Units

[g]

[g]

C

Mean

0.0056

0.0058

SD

0.0017

0.0020

N

10

10

LD

Mean

0.0060

0.0066

SD

0.0014

0.0012

N

10

8

MD

Mean

0.0065

0.0058

SD

0.0019

0.0019

N

8

8

HD

Mean

0.0061

0.0055

SD

0.0010

0.0011

N

8

8

 


 

 

Applicant's summary and conclusion

Conclusions:
There were no treatment related effects on reproduction and development. The NOAEL for reproductive toxicity is 250 mg/kg bw
Executive summary:

In a test according to OECD 422 the substance was administered to male and female Wistar rats at 0, 50, 100 and 250 mg/kg bw (in water) 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. Hematological, clinical biochemistry and urine investigation were performed on selected males and females from each group.

After 14 days of treatment animals were mated (1:1) for a maximum of 14 days. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12.

From 2 pups/litter on day 4 after birth; from all dams and 2 pups /litter at termination on day 13 and from all adults males at termination, blood samples were collected. Blood samples from the day 13 pups and from the adult males were assessed for serum levels for thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis.

The males were sacrificed after completion of the mating period on treatment day 29 and the females along with their pups were sacrificed on post natal day 13. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on high dose and control animals and dead animals. 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.

The results for the parental animals can be found under the section on repeated dose toxicity. There were no treatment related effects in parental animals at any of the doses tested. The NOAEL for parental toxicity is 250 mg/kg bw.

The substance 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 no test item treatment related effects observed on the number of corpora lutea, implantation sites and live pups born, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

There were no test item related effects on the reproductive indices (copulation, fertility and delivery indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 88.89 % was observed in the MD group as compared to 100 % in control group. This decrease was within the standard pregnancy rate of rat i.e.≥80 % and therefore this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.

No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group. In males and females, no statistically significant dose related effect on pup weight, cube root of pup weight on the day of anogenital measurement, absolute and relative anogenital distance in treatment groups when compared to the controls. No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls. No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls or the historical data. No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups.

Based on the absence of effects on reproduction and development, the NOAEL for reproductive toxicity is 250 mg/kg bw.