1,1'-[oxybis(ethyleneoxy)]diethylene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-31 to 2022-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other assay used for intermediate effect derivation

Remarks:

Dose Range Finding Study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2020-2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Range Finder for OECD422

Qualifier:

no guideline followed

Principles of method if other than guideline:

This non-GLP prelimiminary Dose Range-finding study did not follow a specific OECD guideline, but was designes to allow selection of appropriate dose level for the upcoming OECD 422 study.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar Rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models ans Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: Females were nulliparous and non-pregnant.
- Age at study initiation: Young adult rats, 8-9 weeks old at the start
- Weight at study initiation:Males: 223-240 g; females: 144-165 g
- Housing: Rodents will be group housed (2 animals/sex/group per cage). Type II and/or III polycarbonate. Exceptions: During mating (males and females) and females during gestation, lactation and after weaning: Polycarbonate cages Type II and/or III .
- Number of groups: 3 groups; one control and 2 test item-treated groups
- Number of animals: 12 males and 12 females; 4 animals/group/sex; (2 animals/cages) were used in the study. A sufficient number of spare animals were ordered. Animals not used in the study were assigned to the stock colony of the Test Facility after the in-life phase had been ended.
- Diet:Animals will receive ssniff® SM R/M "Autoclavable complete diet for rats and mice .Abreeding and maintenance" produced by ssniff Spezialdiäten GmbH (ad libitum)
- Water: Animals will receive tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1-26.0 ℃ ( target:22 ± 3 °C)
- Humidity (%): 22-53% ( target: 30 - 70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
The test item was formulated in the selected vehicle (corn oil), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic stirrer during the preparation and shortly before the treatment and during the treatment. Prepared formulation should be stored in refrigerated (2°C to 8°C), protected from light. The formulation has eight days stability in corn oil. Formulations was prepared weekly to allow their use according to stability assessment results.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concerning analytics, sampling for homogeneity and concentration control analysis was performed at the start of the administration period and once during gestation as well as lactation. Samples of all concentrations were measured by the Analytical Laboratory of the test facility.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females; 4 animals/group/sex

Control animals:

yes, concurrent vehicle

Details on study design:

The objective of the study was to obtain preliminary information on the toxic potential of the test item administered daily to Han-Wistar rats by oral gavage at three dose levels for 14 consecutive days. The results were helped to determine the dose levels for a subsequent OECD No. 422 study. For this reason, young adult animals were used in this study. Male and female Han-Wistar rats (8-9 weeks old animals) were treated in the study for 14 consecutive days as shown below. The first day of dosing was designated as Day 0 for each animal. Mortality checking and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals on Days 0, 3, 7, 10, 13 and for body weight measurement, prior to scheduled necropsy on Day 14 (fasted). Gross macroscopic examination was performed at necropsy in each animal. Selected organs were weighed, and selected tissues .
were preserved in fixative

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule::General (routine) clinical observations were made daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and daily thereafter. It was performed after treatment (1-2 hours after dosing as no peak period of effects was observed on Day 1 after dosing).

BODY WEIGHT: Yes
- Time schedule for examinations: he body weight of each animal was recorded with a precision of 1 g at randomisation (pre-treatment period, Day -1), on the first day of treatment (Day 0, prior to start of treatment), then on Day 3, on Day 7, on Day 10 and on Day 13 (last treatment day) and prior to necropsy (scheduled necropsy, on Day 14 (fasted)).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The determination of food consumption was performed for all groups on Day 0, then on Day 3, on Day 7, on Day 10 and on Day 13 (last treatment day). The remaining, non-consumed food was weighed with a precision of 1 g.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No water consumption was measured in the study.

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: No ophthalmoscopy was conducted in this DRF study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 14
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 14, clinical pathology investigations.
- Animals fasted: Yes
- How many animals: all

PLASMA/SERUM/LIPIDS: Yes

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

IMMUNOLOGY: Not specified

Sacrifice and pathology:

All animals were euthanized at termination on Day 14. Gross necropsy was performed on each animal. Weight of selected organs (liver,spleen,kidneys,adrenals,brain) was measured, and abnormal organs and tissues were retained. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

Statistics:

At the Test Facility, the statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests were adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared
test was used for statistical differences relative to control.
For pathology data (macroscopic data) the Cochran-Armitage test for trend was applied, then if
appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a
user-defined value (0.05), a pairwise test of each treatment group versus the control group was
made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger
then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as
appropriate.
Note that the small group

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no symptoms observed in the Control groups or the Low dose female group and no
adverse symptoms in Low dose males. Treatment related effects were observed in the High
dose groups for both sexes. Slight salivation was recorded for 1/4 Low dose male on Day 12 and slight to moderate salivation was recorded for 4/4 High dose males on Day 11 and 4/4 High dose females on Day 11 and Day 12. Slightly to extremely decreased activity was recorded for 4/4 High dose males from Day 4 to Day 12 and for 4/4 High dose females from Day 4 to Day 5, at 10 or 15 minutes after dosing; on the occasions where decreased activity persisted after the dosing time, it was generally only slight. The symptoms were seen on 6/14 days for males and 2/14 days for females. Increased salivation was observed 4/4 High dose males and 4/4 High dose females.The
symptom was only on Day 10. Ataxia was observed for 1/4 High dose male on Day 10 only (at 15 minutes after dosing). Piloerection was observed in 2/ 4 High dose males on Day 4 and on Day 6-7, respectively. In summary, a range of symptoms were seen in High dose animals indicating effects that were not life-threatening, with no adverse treatment effects in the Low dose group.

Mortality:

no mortality observed

Description (incidence):

No mortality was recorded in the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no statistically significant effect of test item on body weight or body weight gain in
either sex. At 1000 mg/kg bw/day dose males had an overall weight gain of almost 30% below the controls but the body weight was only about 4% below control at the end of exposure. Other groups had weight values in the normal range.
weight values in the normal range

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear effect on food consumption observed during the study. Statistically significantly decreased food consumption was recorded for High dose males between Day 0-3 and Day 0-7, but with only 2 cages per group the statistical differences are not reliable.

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):

No test item-related effects were seen on the examined haematology parameters in the dosed groups. There was a statistically significantly higher Luc relative (p<0.01) in the 300 mg/kg bw/day female group, but without dose response so it is considered as not test item related effect.There were no other statistically significant or biologically relevant changes in any of the dose.
groups compared to the control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Creatinine (enzymatic) was statistically significantly higher in both dosed male groups (p<0.05)
but without a dose response. Therefore, a compound-related effect is doubtful. Glucose was statistically significantly higher in the High dose male group (p<0.05), but the High dose value was close to the historical control mean for this strain of rat, hence was not considered to be a treatment effect.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Terminal body weights of animals were not significantly different between the groups. There was statistically significantly increased (p<0.05) liver weight (body related) observed in the High dose male group and liver weight (body and brain related) in the High dose female group (p<0.01 and p<0.05). The male and female liver weights adjusted for body weight were ~20% above control. For the High dose female group the body related weight of the liver was outside of the historical control range, therefore it is considered as test item related effect

Key result

Remarks on result:

not measured/tested

Key result

Critical effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD No. 43 Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-[oxybis(ethyleneoxy)]diethylene

EC Number:

212-133-3

EC Name:

1,1'-[oxybis(ethyleneoxy)]diethylene

Cas Number:

764-99-8

Molecular formula:

C8H14O3

IUPAC Name:

{2-[2-(ethenyloxy)ethoxy]ethoxy}ethene

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Hannover Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11/10 weeks old (males/females) at start of the treatment and 13/12 weeks old (males/females) at mating
- Weight at study initiation: (P) Males: 311-363 g; Females: 172-196 g
- Fasting period before study: no
- Housing: group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Type II, III and/or IV polycarbonate cages
- Diet: ad libitum, ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance”
- Water: ad libitum, tap water from the municipal supply
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-25.0℃ (target range: 19-25°C)
- Humidity (%): 36-61% (target range: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (corn oil), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level. The formulations were stirred with magnetic stirrer during the preparation and shortly before the treatment and during the treatment. Prepared formulation was stored in a refrigerator (2°C to 8°C), protected from light for a maximum of 4 days prior to administration to animals according to stability assessment results of the analytical method development and method validation studies.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of a trial formulation, and the Dose Rage Finding (DRF) study and analytical method development study, corn oil was selected as vehicle for this study. Corn oil was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: 20, 62.5, 200 mg/mL
- Amount of vehicle: A constant volume of 4 mL/kg bw was administered to all animals.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days.
- Proof of pregnancy: the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy)
- Further matings after two unsuccessful attempts: no
- 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:

The measured concentrations of the test item in the different formulations varied between 97% and 106% of the nominal concentrations. The results were considered to be acceptable. All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 5 days mating period, then main animals were treated through gestation and up to and including the day before necropsy (13 days postpartum dosing). The day of birth (when parturition is complete) was defined as Day 0 postpartum.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a Dose Range Finding (DRF).In the DRF study (after a 14-day treatment period), at the 1000 mg/kg bw/day dose level in both sexes, transient clinical signs (decreased activity, increased salivation / salivation, piloerection, ataxia) were recorded for the majority of the animals, no similar findings were noted at 300 mg/kg bw/day (only salivation was observed). No test item related clearly adverse effect was seen on body weight or food consumption. No test item-related effects were seen on the examined clinical pathology parameters in the dosed groups. Liver weights adjusted for body weight were ~20% above control in both sexes at 1000 mg/kg bw/day, but there was no evidence at necropsy of any test item-related macroscopic findings in animals dosed at 300 or 1000 mg/kg bw/day. The high dose was set at 800 mg/kg bw/day; lower doses were spaced with a factor of approximately 3. Based on increased liver weights of ~20% after 14 days and taken into account the significantly longer administration period in the present study, the dose level of 1000 mg/kg bw/day was considered as probably too high. Therefore, a high dose of 800 mg/kg bw/day was selected for the present study. The other dose levels were selected to provide a dose-response relationship and a NOAEL at the low dose level.

- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). General (routine) clinical observations were made once a day, during the pre-treatment, treatment in the afternoon (pm). Routine clinical observation was performed twice daily from Day 7. No general clinical observations were made on the day of necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period on Day -7, and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am) or before treatment) and before necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat). Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, then on Day 0, and afterwards weekly, and at termination. Parent females were weighed at least on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0 (i.e. within 24 hours after parturition), 4, 7, 10 and 13, and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g weekly (on body weight measurements day). Food consumption was measured more frequently during the gestation period (at least on GD 0, 3, 7, 10, 14, 17 and 20) and lactation period (at least on PPD 0, 4, 7, 10 and 13).

WATER CONSUMPTION AND COMPOUND INTAKE: No

HAEMATOLOGY AND BLOOD CLOTTING TIMES
Seven male and five female animals from each main group were selected for clinical pathology sampling and analysis at termination (on Day 28 in males, on PPD 14 in females).The following parameters were evaluated in animals selected:
RBC Red Blood Cell (erythrocyte) count, WBC White Blood Cell (leukocyte) count, Hgb Haemoglobin concentration, Hct Haematocrit, MCV Mean Corpuscular (erythrocyte) Volume, MCH Mean Corpuscular (erythrocyte) Haemoglobin, MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, RDW Red Cell (erythrocyte) Distribution Width, Plt Platelet (thrombocyte) count, MPV Mean Platelet Thrombocyte volume, RETIC % Reticulocyte count, NE % Neutrophil, LY % Lymphocyte, MO % Monocyte, BA % Basophil, EO % Eosinophil, LUC % Large Unstained Cells, APTT Activated Partial Thromboplastin Time, PT Prothrombin Time

CLINICAL CHEMISTRY
Seven male and five female animals from each main group were selected for clinical pathology sampling and analysis at termination (on Day 28 in males, on PPD 14 in females). The following parameters were evaluated in animals selected:
Glucose Blood sugar concentration, T-BIL Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ratio, Aspartate Aminotransferase activity, Alanine Aminotransferase activity, Gamma-Glutamyl transferase activity, Alkaline Phosphatase activity, Bile acids

OTHER:
Functional Observational Battery and locomotor activity measurement was performed in the study. Randomly selected 5 males and 5 females per group:
The same animals were used as selected for clinical pathology. Assessment of any potential test item related neurotoxicity was performed during the last exposure week (for example, males on Day 23 am, females on PPD 6-8 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength. In order to avoid hypothermia of pups, dams were normally removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART). A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured. Fore/hind grip strength was measured using a grip strength meter, an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data. Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity. Locomotor activity was monitored by placing each animal individually into a
50 x 50 cm open-field for 1-hour observation time, DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

Oestrous cyclicity (parental animals):

Oestrus cycles was monitored for all females by vaginal smears daily during the pre-exposure period before the treatments starts. Any females that fail to show a 4-5 days cycles were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods). Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle.

Sperm parameters (parental animals):

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

Live pups were weighed individually within 24 hours of parturition (PND0) and on PND4, PND7 and PND13, with accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) was also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND4.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
Animals were euthanized at termination. After blood sampling, gross necropsy was performed on each animal (with special attention to reproductive system and any expected target tissues). Weight of selected organs were measured, and selected organs and tissues were retained.

All animals including the selected animals for blood sampling were fasted (overnight period of food deprivation, in case of females this happened after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) as follows:
- from all dams on PPD 14
- from all non-pregnant adult females on the day of termination,
- from all adult males on the day of termination
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all adult animals was determined:
- with a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and
thymus,
- with a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids.
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight isreported for each animal adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

On completion of the macroscopic examination the following tissues and organs were retained from all adult animals. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

Gross findings, Lungs with bronchi, Skeletal muscle (quadriceps), Adrenal gland, Lymph node, Small intestine, Ovary, Spinal cord, *Aorta, Oviduct, Spleen, Brain, *Pancreas, Sternum with marrow, Cervix, Larynx, *Pharynx, Epididymis, *Pituitary, Stomach, Eye with the optic nerve, Prostate, Testis, *Oesophagus, *Salivary gland (including mandibular, sublingual and parotid glands), Thymus, *Mammary gland, *Nose, *Femur with marrow (with knee joint), Thyroid with parathyroid gland, Heart, Tongue, Kidney, Sciatic nerve, Trachea, Large intestine, Seminal vesicle with coagulating gland, Urinary bladder, *Extraorbital lachrymal gland, Uterus, Harderian gland, Skin, subcutis with mammary gland (inguinal), Vagina

*= no histopathological examination

In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylineosin/phloxine and examined by light microscope. For the adult animals, detailed histological examination was performed as follows:
- on the selected list of retained tissues and organs (as above) in the Control and High dose groups: selected 5 animals/sex/group (the same animals were used for clinical
pathology)
- all macroscopic findings (abnormalities), except of minor order from all animals
- on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina
for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups.

Additional immunohistological staining was performed on all High dose male (12 animals) and all Control male (12 animals) kidneys for alpha 2u globulin and additional histological examination was performed on kidneys of parental males and the stomach (non-glandular) of all remaining parental males and females.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed under anaesthesia on PND4 or/and 13.

For thyroid hormone analysis, blood samples were taken by decapitation into tubes containing K3-EDTA as anticoagulant as follows:
- from up to two pups per litter on PND4,
- from at least two pups per litter on PPD 14 (females) / PND13 (pups)
The collected pup blood (plasma) samples were pooled by litter. The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. At the first instance, samples for the PND13 pups and adult males were assessed for T4 and TSH levels. No additional samples of parental females or PND4 pups were analysed later as it was not deemed necessary.

GROSS NECROPSY
- Dead pups and pups terminated on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. Thyroid gland weight for one male and one female pup per litter were determined. Trimming was done very carefully and only after fixation to avoid tissue damage. No histopathological examination was performed on pups (F1 generation).

Statistics:

In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.
In case of the SAS 9.3 software package the following decision tree was applied for statistical evaluation of continuous numeric data. The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test. For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test.

Reproductive indices:

See Table 1.

Offspring viability indices:

See Table 2.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Control, Low and Mid dose male or female animals.
Slightly decreased activity was observed for 8/12 High dose males from Day 7 to Day 27 and for 8/12 High dose females on Day 16, 18, 27, 33, 40 and 46. The longevity of the symptom was 8 days.
Slight ataxia was observed for 1/12 High dose males on Day 18 and for 5/12 High dose females from Day 16 to 18. The longevity of the symptom was 1 to 3 days.
Slightly to moderately increased salivation was observed for 12/12 High dose males from Day 14 to Day 27 and 12/12 High dose females from Day 15 to Day 54. The longevity of the symptoms was 35 days.
Piloerection was observed for 3/12 High dose males from Day 4 to Day 27. The longevity of the symptom was 3 days.
Recumbency was observed for 4/12 High dose males from Day 7 to Day 21 and 3/12 High dose females from Day 47 to Day 51. The longevity of the symptoms was 4 days.
Red discharge (nose) was observed for 1/12 High dose male on Day 19.
Red coloured urine was observed for 1/12 High dose female on Day 16.
Alopecia was observed for 1/12 Low dose female from Day 34 to Day 50 and for 1/12 Mid dose female from Day 49 to Day 53. The longevity of the symptom was 17 days.
Thin fur was observed for 2/12 Control females from Day 35 to Day 52 or 53, for 3/12 Low dose females from Day 29 to 50 or 51, on Day 53 and for 1/12 Mid dose female on Day 51. The longevity of the symptoms was 1 or 21-22 days.
Decreased activity, ataxia, increased salivation, piloerection, recumbency, red discharge and coloured urine were considered as test item related effects, the others were incidental, not related to the treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant test item related effect on body weight or body weight gain was detected in the study for males. In males, the body weights of the High dose appeared to show lower growth throughout the study (weight growth was ~42% lower than control for D0-27), with the Day 27 body weight ~5% lower than control. Although these differences were not statistically significant, the pattern of growth relative to the pre-treatment period indicates a slight treatment related effect.
Statistically significantly lower (p<0.05) body weight, by about 5%, was observed during the lactation period in the High dose female group, the difference to control is small, but may be a treatment related effect.Statistically significantly decreased body weight gain was observed in the High dose female group between Day7-14 (p<0.05) and Day0-14 (p<0.01), with ~44% lower weight gain to day 14 (similar to the High dose males). The body weight profile of the test item treated animals was comparable with control for the Low and Mid groups, with slightly lower growth in males and females at the High dose, with minor differences occasionally.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect on food consumption was seen in the first two weeks of the treatment in the High dose group (males and females).
Statistically significantly decreased food consumption was observed in the High dose male group (p<0.01 and p<0.05) throughout the treatment period. Data for Day 0-14 period was outside of the historical control range and together with the body weight gain data, it was considered as test item related effect.Statistically significantly decreased food consumption was observed in the High dose female
group during the first two weeks (decreased by -12.6% compared to the Control) (p<0.01 and p<0.05) and during lactation period (decreased by -17.9% compared to the Control) (p<0.01). No such effect was observed during gestation period. The values for High dose females in the first two weeks were out of the historical control range and together with the body weight gain data it was considered as test item related effect.

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):

There were no test item related changes in the male and female dosed group.
Statistically significantly increased neutrophil relative (p<0.01) was observed in the High dose male group when compared to control. Statistically significantly decreased reticulocyte relative (p<0.05) and lymphocyte relative (p<0.01) was observed in the High dose male group when compared to control. The majority of the observed values were within the historical control range, thus these differences were considered as animal variability, not being a test item related effect. No statistically significant and biologically relevant changes were recorded for any other haematology parameters in the test item treated males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item related changes on the serum chemistry in the High dose female group that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in urea concentration was observed in High dose males compared to control. Two of the animals were well above the historical control range therefore, the observed differences were considered as test item related, but in isolation it may not be an adverse effect. Statistically significantly increased calcium level (p<0.05) which appears to be treatment related (and may correspond with kidney histology) increased and bile acid (p<0.01) level was observed in the High dose female group, where the mean value was in the historical control range, but the tendency for high values suggests a treatment related effect.Alkaline phosphatase activity and alanine aminotransferase (ALT/GPT) activity was statistically significantly increased in the High dose female group. The observed data were not considered to be excessively high; the statistical differences are of equivocal relationship with treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item related effects on the T4 and TSH hormone levels and the thyroid gland weight for any of the adult males. The TSH hormone level was measured at the Test Site and later at the Test Facility also to strengthen the outcome of the measurements in case of the male animals. The measurement of the thyroid hormone levels in the adult females, and histology of pup thyroids were not performed as it was not deemed necessary. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
Statistically significantly decreased locomotor activity total distance travelled, was observed for the High dose male group for the first 20 minutes (p<0.01 and p<0.05) compared to the Control, but in the plateau phase (20-60 minutes) the data was not different to the other groups. The other dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was relatively high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. All treated groups had a general profile of activity the same as historical control data. The High dose males had initially lower locomotor activity, but this was no seen at the regular detail observation times in the study, or at the FOB, and females were clearly unaffected, furthermore the profile of the activity over time was fully consistent with normal animals. It is considered doubtful that the statistical differences were a real treatment related effect.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were observed in the kidney in males, and stomach. Immunohistochemical staining for alpha 2 u globulin revealed comparable incidence between Control and High dose male kidneys.

Minimal to mild diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 5/12 High dose males and 4/11 High dose females. Minimal squamous hyperplasia observed in single males from Low and Mid dose males and a 3/11 Mid dose females may be considered within the common background.

Macroscopically, small thymus was observed in 2/12 High dose males and 1/11 High dose females. Microscopically, minimal to mild reduced cellularity of the cortex was observed in the thymus in 1/6 High dose males and 3/5 High dose females. Considering the low incidence of macroscopic and microscopic findings at low severity (minimal mostly) in the thymus and organ weight changes only in high dose males, these changes are considered incidental and not related to test item administration. All other changes seen in Control and/or treated animals, were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedural or a common background.

No microscopic changes were observed in the non-pregnant females.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the different groups) before starting the treatment period. No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 3.98, 4.00, 4.03 and 4.02 days in the Control, Low dose, Mid dose and High dose groups, respectively). Prolonged oestrus was recorded in one case for Mid dose group, this fact was considered as being an occasional finding, not being a test item related effect.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in treated groups (males and females). The fertility index was 92, 100, 92 and 92% in the Control, Low, Mid and High dose groups (males and females). The gestation index was 100% in the Control, Low, Mid and High dose groups. Test item administration was considered to have no impact on the duration of the mating period.
Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 4 days of pairing (cohabitation). The mean duration of mating was 2.67, 2.83, 2.17 and 3.00 days in the Control, Low, Mid and High dose groups, respectively.
There was no effect of treatment noted during the gestation period, parturition and post-partum period in any of dose groups.The mean duration of pregnancy was comparable in the Control and test item treated groups. As far as it could be observed during the study, the parturition was normal for all animals. The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant differences were noted. There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed on reproductive parameters

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or abnormalities were recorded for any pups.
Evidence of suckling was recorded for all live born pups in the study.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation). Statistically significantly decreased number of live born was observed in the High dose group. Statistically significantly increased post-natal mortality (PND0-4), total mortality (PND4), post-natal mortality (PND0-13), total mortality (PND13), number of viable pups (PND0-4), dead pups (born alive PND0-4) and dead pups (born alive PND0-13) was observed in the High dose group. The values were within the historical control range of the rat strain used. Therefore these differences were considered to be incidental. There were no significant differences or effects that could be ascribed to treatment on the prenatal, post-natal or total mortality values (litter mean and %) in the other dose groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related differences in the offspring body weights or weight gains in Low, Mid and High dose groups when compared to the controls. The measured values were within the range commonly recorded for this strain and age. Statistically significantly decreased mean litter pup body weight was observed on PND0 (p<0.05) and PND13 (p<0.05) in the High dose group. Statistically significantly decreased mean litter male body weight on PND7 (p<0.05), on PND13 (p<0.01) and mean litter female body weight on PND13 (p<0.05) was observed in the High dose group. Data were within the historical control range and the dams also had slightly low body weights (-14.2% (p<0.05)), hence these statistical differences alone are not considered to represent a clearly adverse effect of treatment on pup weight.
Statistically significantly decreased mean litter body weight gain was observed between PND7-13 (p<0.05) and PND0-13 (p<0.05) was observed in the High dose group. Statistically significantly decreased mean litter male body weight gain was observed between PND4-7 (p<0.05), PND7-13 (p<0.01), PND0-13 (p<0.01) and mean litter female body weight gain between PND7-13 (p<0.05) was observed in the High dose group. The fact that there was a slightly lower birth weight at the High dose, then the growth rate to termination was ~14% below controls, suggests that there was a test item effect, although the dam weights were affected so the pup weight differences are likely to be related to dam effects. There was no clearly specific effect of the test item directly on pup growth.

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

Description (incidence and severity):

The sex ratio was not effected.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance during the study. No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention during the study. There was no nipples/areolae presence seen in any of the male pups on PND13

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The terminal body weight of the pups was statistically significantly decreased (by -12.4%) in the High dose group and the absolute thyroid weights were also lower, but when adjusted for the body weight, there was no effect on thyroid weights. Also, data were within the historical control range; the statistical difference in absolute weights were not considered as a test item related thyroid effect.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in macroscopically examined pups.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item. The measurement of the thyroid hormone levels in the PND4 pups and histology of pup thyroids were not performed as it was not deemed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no