Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 November 2017 to 24 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Recognized by international guidelines as a recommended test system.
Oral gavage was used as the route of administration as recommended by the OECD 422 guideline, in order to deliver accurate doses.

Test material

Constituent 1
Chemical structure
Reference substance name:
Castor oil, hydrogenated, ethoxylated
EC Number:
500-147-5
EC Name:
Castor oil, hydrogenated, ethoxylated
Cas Number:
61788-85-0
Molecular formula:
C57H110O9 (C2H4O)n where n = 1-6.5
IUPAC Name:
Castor oil, hydrogenated, ethoxylated
Test material form:
solid
Remarks:
pasty wax

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recognized by international guidelines as a recommended test system
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Hannover Wistar Rat (HsdHan®:WIST)
- Source: Envigo RMS B.V. Kreuzelweg 53 5961 NM Horst Netherlands
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 291-386 g;Females: 197-240 g
- Fasting period before study: no
- Housing: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating (maximum 5 animals/cage) Makrolon type-IV cages
Mating (one male and one female/cage) Makrolon type-III cages
Postmating, gestation and lactation (individual) Makrolon type-III cages
- Diet: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (during lactation Teklad 2018C rat/mouse maintenance diet)
- Water: tap water ad libitum
- Acclimation period: 5 days and 15 days pre-test

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24ºC:
- Humidity: 40-70 %
- Air changes (per hr): 15-20 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily (stored at ambient temperature and in the dark)
1.The necessary quantity of test item was weighed in a single-use container.
2.Small amounts of vehicle were added (approximately 60-70% of the total volume) and mixed with a manual rod. Any lumps were broken up at this point and the final result was an oily solution.
3.The oily solution was transferred to a volumetric flask or graduated measuring cylinder that has been previously moistened with the vehicle. The mortar and the single-use container were rinsed with the vehicle to ensure that there were no remnants of the test item. This vehicle was added to the volumetric container and made up to the mark.
4.When it was necessary to adjust volume, the volume was first added into volumetric vessel and after the remainder was passed at final container. Before the formulation was passed to the final container, it was assured that the formulation was homogenous, and when it was necessary a vortex was used for this purpose.
5.The oily solution was transferred to a suitable container and submitted to stirring for overnight before aliquoting for analysis when necessary.

VEHICLE: propylene glycol
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 4 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration on day 1 and in week 3 were analyzed for achieved concentration of the test item. Prior to initial sampling each day, the formulation was mixed by 20-fold inversion and paddle stirring for minimum of 20 minutes. A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of propan-2-ol. The extract was diluted, where necessary, using propan-2-ol, to provide a solution containing the substance at an expected concentration within the range 200 µg/mL to 400 µg/mL. The concentration in the final solution was quantified by HPLC using evaporative light scattering detection.

Method:
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and an
Alltech Model 3300 ELSD detector
Column: Phenomenex Jupiter C18, 300 Å, 5 µm, 250 × 4.6 mm
Column temperature: 50ºC
Sample temperature: Ambient
Mobile Phase: Propan-2-ol
Flow rate: 1 mL/min
Needle wash: Propan-2-ol
Injection volume: 20 µL
Auto sampler run time: 7 minutes
Empower run time: 6 minutes
Approximate retention time: 3.3 minutes

Detector settings:
Drift tube temperature: 80ºC
Nitrogen flow: 1.5 L/min
Gain: 2

e-SATIN settings:
Environment: 60 Hz
Enable BCD: Off
BCD polarity: positive
Rate: 5 points/second
Description: Channel 1
Scale factor: 1000
Units: mV

Results method validation:
Calibration linearity (range 100-500 ug/mL): r > 0.999 (quadratic relationship)
Specificity: absence of peak for substance in control sample
Precisions calibration (CV 0.97% 100 ug/mL, 0.09% 500 ug/mL)
Accuracy: procedural recovery value of 92.1% (CV=0.83%, n=5) was obtained for 1 mg/mL and 100.2% (CV=0.36%, n=5) was obtained for 200 mg/mL.
LOQ: 1.11 µg/mL and 3.17 µg/mL (3 and 10 times baseline noise)

Preparation analyses:
Homogeneity CV 0.63-2.30% at 20 mg/mL; 0.14-2.78% at 200 mg/mL
Stability over 8 days at 21 °C: 83.4% of initial at 20 mg/mL; 94% of initial at 200 mg/mL
Stability over 15 days at 4 °C: 82.5% of initial at 20 mg/mL; 97.1% of initial at 200 mg/mL
Procedural recovery during test runs: 89.2-103.1% at 20 and 200 mg/mL
Accuracy: 67-107% of nominal 20 mg/mL; 84-106% of nominal at 60 mg/mL; 80-104% of nominal at 200 mg/mL

Day 1 samples at 20 mg/mL were outside the required purity range, as the samples taken in week 3 were within the purity ranges and it concerns the lowest concentration only, this has not affected the study results.
Duration of treatment / exposure:
Males Two weeks pre-pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 13/15 of lactation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
measured concentration at day 1 67% of nominal
measured concentration in week 3 107% of nominal
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
measured concentration 84-107% of nominal
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
measured concentration 80-104% of nominal
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on the results of a 2-week dose range finding study
In that study (Hannover Wistar rats dosed at 0 (PEG), 100, 500 or 1000 mg/kg/day) there were no mortalities or substance related clinical signs observed. Food consumption, body weight or water consumption were considered to be unaffected by treatment. There were no substance related macroscopic findings or effects on organ weights. Under the conditions of this study, the daily oral gavage administration of the substance daily for 14 consecutive days at dose levels up to 1000 mg/kg/day caused no signs of evident toxicity

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly (twice pre-treatment)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in a standard arena before treatment commenced and during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males Weekly during pre-treatment
On day 1 and weekly thereafter.
On the day of necrospy.
F0 females Weekly during pre-treatment
On day 1 and weekly thereafter
Days 0, 7, 14 and 20 after mating.
Day 1, 4 and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION : Yes
- Time schedule for examinations: weekly except during mating

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes 6-8 hours
- How many animals: 5/sex/group
- Parameters checked: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count:, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (SPT) and Activated partial thromboplastin time (SAPT)
For samples showing abnormalities in the automated analysis a blood film was analysed microscopically for differential leucocyte count (2 females at 0 mg/kg/day, 1 female at 100 mg/kg/day, 2 females at 300 mg/kg/day and 1 female at 1000 mg/kg/day).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes 6-8 hours
- How many animals: 5/sex/group
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Creatine kinasa (CK), Glucose (Gluc), Total cholesterol (Chol), HDL, LDL, Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Protein electrophoretogram

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males in week 5, females at day 7-9 of lactation
- Dose groups that were examined: 5/sex/group
- Battery of functions tested: sensory activity (includes: Blink reflex, Pinna reflex, Iridic light / Pupil closure reflex, Proprioception (right leg) / push-off (hind legs), Pain response / Tail pinch response, Auditory Startle /hearing reflex, Righting reflex in the air), grip strength (fore-and hind limb) and motor activity(beam crossing over 10 min intervals for 1 hour)

IMMUNOLOGY: Yes
- Time schedule for examinations: at termination
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes 6-8 hours
- How many animals: all adult males
- Dose groups that were examined: all
- Parameters checked: serum samples of the adult males for thyroxine (T4) levels (additional taken
samples from all adult females were not further examined)
Oestrous cyclicity (parental animals):
vaginal smears using inoculation loops
-For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
-Daily from the beginning of treatment period until evidence of mating.
-On day of sacrifice.
Sperm parameters (parental animals):
Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
Clinical observations Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size Daily from Day 1-13 of age
Sex ratio Days 1, 4, 7 and 13 of age
Individual offspring body weights Days 1, 4, 7 and 13 of age
Ano-genital distance Day 1 - all F1 offspring
Nipple/areolae count Day 13 of age - male offspring.
Blood sampling for thyroxine analysis:
Day 4 of age Offspring: 2 females per litter (where possible):
Day 13 of age Offspring: 2 males and 2 females per litter (where possible):

Postmortem examinations (parental animals):
ORGAN WEIGHTS: Yes (see tables)

GROSS PATHOLOGY: Yes (see tables)
Males: after final investigations completed (after 5 weeks of treatment)
Non-pregnant females: Day 25-26 after mating
Pregnant females: Day 14-16 of lactation

HISTOPATHOLOGY: Yes (see tables)
Premature deaths All F0 animals from all groups.
Scheduled kill 5 F0 animals in Groups 1 and 4:
All F0 animals. Abnormalities only.
Other examinations
Ovaries: qualitative evaluation of one section from each ovary.

Postmortem examinations (offspring):
External examination with an assessment of stomach for milk content
Statistics:
The following comparisons were performed: Group 1 vs. 2, 3 and 4
For categorical data, the proportion of animals was analyzed for each treated group (as appropriate) versus the control group.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there was evidence against a monotonic dose-response when Dunnett's test was performed instead.
For organ weight data an ano-genital distance (using the litter average pup body weight as covariate), analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.
For estrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups, using scores appropriate to the severity of the observation.
For pre-coital intervals an exact one-tailed (upper-tail) Linear-by-linear test was applied to all groups, using appropriate scores. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05).
For gestation length an exact two-tailed Linear-by-linear test, with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05).
For number mating, number conceiving, number fertile, number live and terminal smear status an exact one-tailed (lower-tail) Cochran-Armitage test was applied to all groups.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Reproductive indices:
Conception rate Number animals achieving pregnancy x 100/ Animals mated
Fertility index Number animals achieving pregnancy x 100/ Animals paired
Gestation index Number of live litters born x 100/ Number pregnant
Offspring viability indices:
Post-implantation survival index Total number offspring bornx 100 / Total number uterine implantation sites
Live birth index Number live offspring on Day 1 after littering x 100/ Total number of offspring born
Viability index (Viability index Day 4) Number live offspring on Day 4 x 100/ Number live offspring on Day 1 after littering
Lactation index (Viability index Day 13) Number live offspring on Day 13 after littering x 100 / Number live offspring on Day 4(after
selection for thyroid hormone bleed)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
no effects in surviving animals
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 male at 300 mg/kg bw was sacrificed for welfare reasons on treatment day 8. It showed the following clinical signs on the day it was sacrificed: reddish nose discharge, prostration, reduced body tone and pallor. Reddish discharge in nose and right dilated kidney pelvis were recorded during necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
males: up to 6% decreased between treatment and post-mating day 15 (not considered treatment related)
females: no treatment related effects
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
males: 89-100% of controls (not considered treatrment related)
females: 67-104% of controls (not considered treatrment related)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: sign decreased leucocytes (lymphocytes) at all doses, no dose response relationship and values within historical control values; decreased APT at 300 and 1000 mg/kg bw (no dose response and non-significant)
Females: increased PT and APT at all doses (no dose response see table); sign increased lymphocytes at 300 mg/kg bw (part of the samples were analysed manually due to errors in the automated assessment)

see overview table
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: sign increased albumin at all doses, no dose response relationship and values within historical control values; effects on Na, K and Cl at all dose groups (no dose response and within historical controls, see table); decreased bile acids at 100 and 300 mg/kg bw
Females:sign decreased creatine kinase at 300 and 1000 mg/kg bw (no dose response and within historical controls, see table); incidental non-sign increase of bile acids and triglycerides

see overview table
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations and grip strength: no treatment related effects
Motor activity: in males and females at 1000 mg/kg/day occasional statistically significant differences compared to controls were observed. Overall effects were non-significant, were considered not to have any toxicological relevancy and were attributed to the normal variability
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The microscopic examination performed 5-8 weeks after treatment revealed no changes related to the substance at 1000 mg/kg/day. All the histological findings were considered to be incidental.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stagespecific abnormalities were noted in males treated at 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
no treatment related effect (oestrus cycle 4-5 days pre-treatment, at termination >90% of females were in diestrous phase)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stagespecific abnormalities were noted in males treated at 1000 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance was not affected by treatment (100% for all groups) and conception and fertility were 80% (8/10) for males and females at 0 and 300 mg/kg/day, 100% for males and females at 100 mg/kg/day and 90% (9/10) for males and females at 1000 mg/kg/day, demonstrating that there was no dose related effect.
There was no substance-related effect on gestation length or gestation index.

Details on results (P0)

The slight effects on body weight and food consumption are considered to be non-adverse. The behavior of the animals in the arena was not affected by treatment. Sensory reactivity, grip strength and motor activity were also unaffected by treatment. The findings in haematology and clinical chemistry were either incidental or within historical control values. There was no relationship with the applied dose. No macroscopic findings were reported and organ weights did not differ from controls. Hisopatholgical examnination revealed noabnormalities. Thre were no effects on the male and female reproductive system. Mating performance, conception,and fertility were was not affected by treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no toxicologically relevant effect of treatment on mean body weights of male or female offspring on day 1 of age or on subsequent body weight measurements until day 13.
High values measured in female pups on day 4 in all treatment groups were considered within normal variance
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:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
no effects on the ano-genital distance of male and female offspring or on male nipple observations.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Details on results (F1)

There was no effect on offspring growth. There were no offspring clinical or necropsy signs indicative of a reaction to the substance. Also, there was no effect on litter size, sex ratio, ano-genital distance or nipple areolae. At 1000 mg/kg/day mean body weight in females’ offspring was increased on day 4 with respect to Control, that seems to be normalized in time, and consequently not considered to be adverse.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Parental effects

Dose (mg/kg bw)

0

 

100

 

300

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

0/10

0/10

0/10

0/10

1/10

0/10

0/10

0/10

No

Clinical signs

NTRE*

No

Body weight (gain)

NTRE

No

Food consumption

NTRE

No

Behavioral effects (wk 5 +lact)

NTRE

No

Motoractivity (wk 5 + lact)

 

 

↑(19% ns)

 

↑(41% ns)

↑ (24% ns)

↑ (25% ns)

↑(13% ns)

No (no dose-response)

Estrus cycle (pre-test +PM)#

NTRE (4-5 days)

No

Pre-coital interval

NTRE (1-4 days)

No

Conception rate (%)

 

No

 

100

 

80

 

90

No

Fertility index (%)

 

No

 

100

 

80

 

90

No

Mating performance

NTRE (100%)

No

Gestation length

NTRE (20-22 days)

No

Gestation index (%)

NTRE (100%)

No

Implementation sites

 

No

 

13.6

 

14.0

 

12.9

No

Litter size (Day 1)

 

No

 

11.8

 

12.8

 

12.9

No

Haematology

 

 

WBC↓ (29%)

Lymph ↓ (34%)

 

 

PT ↑ (10% ns)

APT↑(27% ns)

WBC↓ (24%)

Lymph↓ (28%)

APT↓ (13% ns)

Lymph ↑ (83%)$

PT ↑ (9% ns)

APT↑ (22% ns)

WBC↓ (28%)

Lymph ↓ (31%)

APT↓ (14% ns)

PT ↑ (10%)

APT↑ (59%)

No (within historical controls)

Clinical biochemistry

 

 

Alb↑ (10%)

Na/K/Cl

Bile ac

(137% ns)

Bile ac↓(27% ns)

Alb↑ (17%)

Na/K/Cl

CK↓ (67%)

 

Bile ac↓(32% ns)

Alb↑ (17%)

Na/K/Cl

CK↓ (63%)

Bile ac ↑ (115% ns)

Triglyc↑ (60% ns)

No (within historical controls)

Organ weights

NTRE

No

Marcoscopy

NTRE

No

Histopathology*

NTRE

No

Testes

NTRE

No

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

*in the male that died at 300 mg/kg bw: red nose, reduced body tone, prostrate and whole body pallor on the day of death (day8); unilateral pelvic dilation, in the testes, minimal bilateral tubular vacuolation was seen, along with minimal cell debris and minimally reduced sperm content in the epididymides.

# at termination 90% females in treatment groups were in diestrous phase

$ measured in limited number of animals, but effect stays when manual screening for differential leucocytes is taken into account

Effects in offspring

Dose (mg/kg bw)

0

 

100

 

300

 

1000

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Litter size day 1 (day 13)

13.4 (10.6)

11.8 (9.9)

12.8 (10.9)

12.9 (10.9)

No

BW gain (day 1-13)

20.9

20.5

22.9

22.4

21.2

20.2

21.4

21.1

 

Sex ratio (% M)

52.9

46.3

47.3

56.5

 

Live birth index (%)

100

93.0

97.3

100

 

Viability index (% day 4)

98.0

99.3

100

99.2

 

Lactation index (% day 13)

95.0

100

100

100

 

Anogenital distance (mm)

3.008

1.303

3.082

1.269

3.186

1.231

3.121

1.333

 

Nipples

0/8

 

0/10

 

0/8

 

0/9

 

 

↑/↓= significantly increased/decreased

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on litter size, sex ratio, clinical signs, ano-genital distances or macroscopy.
Executive summary:

In this study according to OECD 422, oral administration (by gavage) of the substance to Wistar rats at the doses of 100, 300 and 1000 mg/kg/day for two weeks prior to mating and up to the day before sacrifice inclusive (males) or up to days 13-15 of lactation (females) was well tolerated.

No test item related mortality was recorded during the study.

There were no signs of evident toxicity related to clinical signs, sensory reactivity, grip strength or motor activity. Regarding body weights and food consumption, lower mean values with respect to Control were recorded in males as well as for food consumption in females. However, given the magnitude observed and the fact that the effect in food consumption was also recorded during pre-test, it needs to be considered as a non-adverse effect.

The statistical differences observed in in hematology, coagulation or clinical biochemistry were not considered to be test item related, based on the magnitude and in the absence of a dose relation. These values were within those observed in rats of this strain and age and were there attributed to normal biological variation.

There was no indication of an effect of the substance on T4 levels and there was no evidence of a test-item effect in the histopathology performed on F0 adults.

There were no changes in the macroscopic examination or organ weights that could be attributable to the substance.

Administration of the substance to Wistar rats by oral gavage for 5-8 weeks was not associated with macro/micropathological findings in this study. In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in males treated at 1000 mg/kg.

The NOAEL for repeated dose toxicity was considered to be 1000 mg/kg/day for males and females, taking into account the slight decrease in food consumption and body weights.

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in males treated at 1000 mg/kg.

Estrous cycles and reproductive parameters of pre-coital interval, mating performance, fertility or gestation length or index were unaffected by treatment.

There was no effect on offspring growth. There were no offspring clinical or necropsy signs indicative of a reaction to the substance. Also, there was no effect on litter size, sex ratio, ano-genital distance or nipple areolae. At 1000 mg/kg/day, there was a slight reduction in the offspring survival index on lactation day 13 as well as mean body weight in females’ offspring was increased on day 4 with respect to Control, that seems to be normalized in time, and consequently not considered to be adverse. 

The NOAEL for reproductive/developmental toxicity is 1000 mg/kg/day.