Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 September 2014 and 13 April 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Some procedures were performed on Day 25 or 26 post coitum rather than post partum, as the group failed to achieve pregnancy.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for at
least six days during which time their health status was assessed. A total of ninety seven animals
(forty eight males and forty nine females(including one replacement animal)) were accepted into
the study. At the start of treatment the males weighed 296 to 346g, the females weighed 196 to
229g, and were approximately twelve weeks old.
Initially, all animals were housed in groups of three in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group. Following
evidence of successful mating, the males were returned to their original cages. Mated females
were housed individually during gestation and lactation in solid floor polypropylene cages with
stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during gestation and lactation.
Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
The diet, drinking water, bedding and environmental enrichment was considered not to contain
any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. There were no deviations from the target
range for temperature and transient deviations from the target range for relative humidity were
considered not to have affected the purpose or integrity of the study; see deviations from Study
Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study.
Results show the formulations to be stable for at least seventeen days in the dark at approximately 4 °C. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.
Samples of the test item formulation were taken and analyzed for concentration of 1,3- Propanediol, 2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with (chloromethyl) oxirane CAS RN: 30499-70-8 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 94% to 107% of the nominal concentration confirming the accuracy and suitability of the formulation procedure.

One female (37R) at 30mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen right eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Details on mating procedure:

On Day 15 (Day 16 for male 25 and female 37), animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC / UV) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot, 250 mg of test item was accurately weighed into a 2500 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 0.1 mg/L. Aliquots of this stock standard solution were used to prepare working standard solutions in water with a concentration of 0.001 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then shaken to dissolve. Where necessary, sample solutions were further diluted with acetonitrile (intermediate dilutions) and then water (final dilutions) to achieve the working concentration.

Preparation of accuracy samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Instrument set up
HPLC system: Agilent Technologies 1200 MSD, incorporating autosampler and work station
Mass selective detector
Source: Electrospray
Fragmentation energy: 90 100, 150 and 160 volts
Polarity: positive
Mode: single ion mode with 247.1, 269.2. 320.3, 571.2 amu
Gas temperature: 350 °C
Drying gas: 11.1 L/min
Nebuliser pressure: 40 psi
Capillary voltage: 1500 volts
Gain: 1
Column: Luna C18, 5 µ (150 x 2 mm id)
Column temperature: 40 °C
Gradient elution:
Eluent A: 0.1 % formic acid in LC-MS water
Eluent B: 0.1 % formic acid in acetonitrile

Time (mins) %A %B
0 80 20
5 0 100
10 0 100

Flow rate: 0.4 mL/min
Injection volume: 10 µL
Retention time: Approximately 5 to 7 minutes

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Results of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method and retention time was confirmed.

Linearity
The data was found to have a quadratic correlation within the calibration range of 0.005 to 0.0015 mg/L. The R2 fit of the calibration curve to the data was 0.996 and was considered to be acceptable.

Accuracy
The fortified samples of PEG 400 were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 94 – 107 % and, thus, the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 17 days in the refrigerator (4 °C).
In conclusion, the results indicate the accurate use of the test item and PEG 400 as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable correlation to the fit of a quadratic equation. The analytical procedure was found to have acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.

Duration of treatment / exposure:

Up to 56 days

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study
One female (37R) at 30mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study. The replacement female started one day behind the other animals so was paired for mating one day later than the other females on the study.

i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15 (Day 16 for male 25 and female 37), animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli. As no littering females were available at 300 mg/kg bw/day, females at this dosage were assessed on Day 25 post coitum.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. As no littering females were available at 300 mg/kg bw/day, females at this dosage were assessed on Day 25 post coitum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Females at 300 mg/kg bw/day were terminated on Day 26 post coitum.

Remarks:

Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Twelve per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Positive control:

None

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and approximately one hour after dosing. All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.


Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:

Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

One female (37R) at 30 mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen right eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study. The replacement female was placed in the same cage as the original female. As the cage always contained the correct number of animals it was considered acceptable to continue with the food consumption for this cage and use it in the assessment of effects during the first week of the study although the initial food intake would have included a contribution from the excluded animal.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females, Day 25 post coitum for females receiving 300 mg/kg bw/day). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Postmortem examinations (parental animals):

Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum and Day 25 post coitum for females at 300 mg/kg bw/day. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). Where possible the corpora lutea were also counted.

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group:

Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

Tissues shown below were weighed from all remaining animals:
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

As all females were non-pregnant at 300 mg/kg bw/day, normal range data based on 90 Day Toxicity studies are also present for female (non-pregnant) animals.


Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:

Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes•, Liver, Thymus, Lungs (with bronchi) #, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina.

* = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 300 mg/kg bw/day dose group animals, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in below from the remaining control and 300 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. Female 37R was excluded from histopathological processing and investigation. In addition, sections of testes from all control and 300 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Testes, Uterus/Cervix, Vagina, Mammary gland

Detailed qualitative examination of the testes was undertaken, 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, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

As there were no indications of treatment-related changes, examination was not extended to include animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (Jeffrey Wilson at Propath GmbH, Muttenzerstrasse 30, 4133 Pratteln, Switzerland). A peer review of findings was conducted by the Test Facility.

Postmortem examinations (offspring):

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

See below

Reproductive indices:

See below

Offspring viability indices:

See below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See results

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

See results

Mortality
There were no unscheduled deaths on the study that were attributed to treatment.

One female (37R) at 30 mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study.

Clinical Observations
Clinical signs associated with treatment during the study were restricted to increased post-dosing salivation for all males and, to a lesser extent, the majority of females at 300 mg/kg bw/day. Increased post-dosing salivation is frequently observed when a slightly unpalatable or irritant test item is administered via the oral gavage route and is generally considered to reflect distaste of the dosing formulations rather than any adverse systemic effect of treatment.

At 100 mg/kg bw/day, one male showed incidences of pilo-erection on two separate occasions during the study, but in the absence of any similar findings at 300 mg/kg bw/day, this isolated finding was considered to be incidental and unrelated to treatment.

No clinical signs were apparent during the study for both sexes at 30 mg/kg bw/day or for females at 100 mg/kg bw/day.


Functional Observations
There were no abnormal observations apparent during behavioural assessments and there was no consistent pattern in intergroup differences for behavioural assessment scores that indicated any effect of treatment.

Functional Performance Tests
There were no changes in functional performance that were considered to be related to treatment.

For females at 30 mg/kg bw/day, lower mean values for the last 20% mobile activity attained statistical significance when compared with control but, in the absence of any similar effect at higher dosages this finding was considered to be incidental and unrelated to treatment.

Sensory Reactivity Assessments
Inter-group differences in sensory reactivity scores did not indicate any effect of treatment for either sex at 30, 100 or 300 mg/kg bw/day.


Body Weight
Body weight gain of males was considered to be unaffected by treatment throughout the study at 30, 100 and 300 mg/kg bw/day.

At 300 mg/kg bw/day, body weight gain of males was slightly lower than control during the final week of the study with differences attaining statistical significance. Group mean body weights are influenced by the laboratory investigations being performed during this period and, as body weight gains prior to this period, were essentially similar to control, these differences were considered to reflect normal biological variation rather than any treatment related effect of the Test Item.

Body weight gain of females was considered to be unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Body weight gain of females during pregnancy and lactation was considered to be unaffected by treatment at 30 and 100 mg/kg bw/day. Body weight gain during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.


Food Consumption
At 300 mg/kg bw/day, food consumption for males was lower than control during the first week of treatment; thereafter food consumption was essentially similar to control and was unaffected by treatment.

There was no obvious effect on food consumption for males at 30 and 100 mg/kg bw/day.

Food consumption of females was considered to be unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Food consumption of females during pregnancy and lactation was considered to be unaffected by treatment at 30 and 100 mg/kg bw/day. Food consumption during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.


Food Conversion Efficiency
Food conversion efficiency for both sexes during the pre-pairing phase and males during the post-mating phase of the study was considered to be unaffected by treatment at 30, 100 and 300 mg/kg bw/day.


Water Consumption
Visual assessment of water consumption during the study did not indicate any obvious effect of treatment on water intake for either sex at 30, 100 or 300 mg/kg bw/day.

Reproductive Performance
Mating and Fertility
There were no treatment-related effects on mating performance as assessed by pre-coital interval, with all treated animals mating within the first four days of pairing (i.e. at the first estrus opportunity); however at 300 mg/kg bw/day none of the matings resulted in a pregnancy.


Gestation Length
There was a tendency for gestation length to be longer than control for females receiving 30 or 100 mg/kg bw/day with differences from control attaining statistical significance: however all gestation lengths were within the normally expected range observed within this laboratory for this strain of rat.

Laboratory Investigations
When assessing the results of laboratory investigations for females at 300 mg/kg bw/day, it has to be considered that all these females were non-pregnant and were therefore in a different physiological state to the remaining females on the study.


Hematology
Intergroup differences for a number of haematology parameters for females at 30 and 100 mg/kg bw/day and both sexes at 300 mg/kg bw/day attained statistical significance when compared with control but none of these findings were supported by any histopathological change and, at the levels observed, these were considered not to indicate any adverse effect of treatment.

For males at 300 mg/kg bw/day, higher erythrocyte counts and haemoglobin levels attained statistical significance but all individual values for these treated animals were within the historical control range.

For females at 300 mg/kg bw/day, higher erythrocyte count, hemoglobin and hematocrit level and lower mean cell hemoglobin and mean cell volume attained statistical significance when compared with control. With the exception of one value for erythrocyte count and hemoglobin, values for these treated animals were within the historical control range, while for the control females, one value for erythrocyte count, mean cell hemoglobin and mean cell volume were outside this historical range. Statistically significantly lower mean cell volume was also observed for females at 30 and 100 mg/kg bw/day but all individual values were within the historical control range.

For females at all dosages, total leukocyte count and the number of neutrophils were statistically significantly lower than control; all values for these treated animals were within the historical control range, while one control value for total leukocyte count and two control values for neutrophils were outside this historical range. At 300 mg/kg bw/day, higher numbers of lymphocytes attained statistical significance when compared with control but only one value for these treated animals exceeded the historical control range.

For females at 100 and 300 mg/kg bw/day, lower platelet counts attained statistical significance compared to control; all individual values for these treated animals were within the historical control range but one control value exceeded this historical range.

For females at 30 mg/kg bw/day, there was a statistically significant increase in prothrombin time compared to control; there was no similar increase at higher dosages and all individual values for these treated animals were within the historical control range.

Blood Chemistry
Intergroup differences for a number of blood chemistry parameters for females at 30 and 100 mg/kg bw/day and both sexes at 300 mg/kg bw/day attained statistical significance when compared with control but none of these findings were supported by any histopathological change and, at the levels observed, these were considered not to indicate any adverse effect of treatment.

For males at 300 mg/kg bw/day, higher creatinine levels attained statistical significance compared to control; this was principally due to one treated animal with a particularly high recorded creatinine level, all other values were within the historical control range.

For females at all dosages, albumin/globulin ratio was statistically significantly higher than control but there was no consistent dosage relationship. Additionally, there were no accompanying statistically significant changes in the levels of total protein or albumin and all values for albumin/globulin ratio within the historical control range.

For females at 300 mg/kg bw/day, higher glucose, calcium and inorganic phosphorus levels attained statistical significance compared to control; two glucose values and inorganic phosphorus values exceeded the historical control, all other values were within this historical range.

For females at 300 mg/kg bw/day, lower alanine aminotransferase and alkaline phosphatase levels attained statistical significance compared to control; all values were within the historical control and a decrease in these parameters is unlikely to indicate an adverse effect of treatment.

Necropsy
Adults
Neither the type, incidence or distribution of macroscopic findings apparent at terminal necropsy indicated any obvious effect of treatment at 30, 100 or 300 mg/kg bw/day.
Organ Weights
At 300 mg/kg bw/day, there was a decrease in absolute and body weight relative liver weights for females compared with control, with differences attaining statistical significance. These differences are considered to reflect the difference in pregnancy state between the pregnant controls and the non-pregnant treated females. Body weight relative values are considered to be the best indicator of toxicological effect for this organ and all body weight values for these treated animals were within the historical range based on Ninety Day Toxicity studies where females are of similar age and the same pregnancy status.

At 300 mg/kg bw/day, there was an increase in absolute and body weight relative thymus weights for females compared with control, with differences attaining statistical significance. Again, these differences were probably influenced by the difference in pregnancy state between the pregnant controls and the non-pregnant treated females; all individual thymus weights were within the historical range based on Ninety Day Toxicity studies.

At 300 mg/kg bw/day, statistically significantly lower absolute and body weight-relative heart weights, compared to control, were apparent but all values for treated animals were within the historical control range.


Histopathology
Microscopic examination of tissues for both sexes at 300 mg/kg bw/day did not reveal any microscopic alterations indicative of toxicity. There was no evidence of histopathological changes that could give an explanation for the failure of females to achieved pregnancy at this dosage.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There was a clear effect on reproduction leading to no pregnant animals being available for assessment.

Remarks on result:

not measured/tested

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

See results

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

As previously discussed, all females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it is not possible to assess litter responses at this dosage. All control females and females receiving 30 or 100 mg/kg bw/day achieved pregnancy, gave birth and successfully maintained a litter to Day 4 of lactation.


Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30 or 100 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.


Offspring Growth and Development
There was no adverse effect on offspring bodyweight and litter weights at Day 1 and subsequently body weight gain to 4 post partum at 30 or 100 mg/kg bw/day. At 100 mg/kg bw/day, offspring body weights were slightly higher than control on Day 1 of age, with differences for males attaining statistical significance. These differences were considered to reflect the slightly longer gestation period observed for parent females at this dosage rather than any treatment related effect on pre-natal/early post-natal growth.

Offspring performance during assessment of surface righting appeared to be unaffected by maternal treatment at 30 or 100 mg/kg bw/day.

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 30 or 100 mg/kg bw/day.

Necropsy
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal treatment at 30 or 100 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

gross pathology

other: offspring litter size / sex ratio / Offspring growth and development / Necropsy

Reproductive effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. There was a clear effect on reproduction at this dosage leading to no pregnant animals being available for assessment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 300 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period. Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Surviving adult males were terminated on Day 43 or Day 44, with the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results…….

There were no adult deaths during the study that were attributed to treatment. Clinical observations identified an increase

salivation post-dosing for all males and the majority of females at 300 mg/kg bw/day.

 

There were no treatment-related changes in the body weight, consumption, behavioural parameters, functional performance or sensory reactivity at 30, 100 or 300 mg/kg bw/day. Body weight gain of females was unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Body weight gain of females during pregnancy and lactation was unaffected by treatment at 30 and 100 mg/kg bw/day. Body weight gain during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.

 

Food consumption of females was unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Food consumption of females during pregnancy and lactation was unaffected by treatment at 30 and 100 mg/kg bw/day. Food intake during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.

Pre-coital interval was unaffected by treatment study at 30, 100 and 300 mg/kg bw/day, but at 300 mg/kg bw/day none of the matings resulted in a pregnancy. There was a tendency towards longer gestation length for females at 30 and 100 mg/kg bw/day.

All females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it is not possible to assess litter responses at this dosage. All control females and females receiving 30 or 100 mg/kg bw/day achieved pregnancy, gave birth and successfully maintained a litter to Day 4 of lactation. 

 

Corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size, sex ratio and offspring survival to Day 4 of age was unaffected by maternal treatment at 30 or 100 mg/kg bw/day. There was no adverse effect on treatment on offspring bodyweight and body weight gain, litter weights, clinical signs, performance of surface righting or necropsy findings at 30 and 100 mg/kg bw/day.

 

There was no adverse effect of treatment on hematology or blood chemistry parameters for both sexes at 30, 100 or 300 mg/kg bw/day. No statistically significant differences were observed for organ weights that were considered to indicate an adverse effect of treatment. Macroscopic necropsy findings did not indicate any effect of treatment for either sex at 30, 100 or 300 mg/kg bw/day. Microscopic examination of tissues for both sexes at 300 mg/kg bw/day did not reveal any microscopic alterations indicative of toxicity or any evidence of histopathological change that could give an explanation for the failure of females to achieved pregnancy at this dosage.

 

Conclusion

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. There was a clear effect on reproduction at this dosage leading to no pregnant animals being available for assessment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant and conducted in accordance with OECD test guidelines

Additional information

An OECD 422 Combined Repeat Dose Toxicity Screening Study was conducted to investigate the potential for adverse effects on systemic toxicity, reproductive toxicity and offspring development. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. However there was a clear effect on reproduction at this dose level, leading to no pregnant animals or offspring. All females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it was not possible to assess litter responses at this dosage. Given these effects, a further investigative study was conducted. 

The investigative study paired treated males with untreated females, and treated females with untreated males. There was an apparent effect on treated male fertility with all mated control females (paired with treated males) failing to achieve pregnancy compared to only two mated treated females (paired with control males). Treatment of the present substance at a dose level of 300 mg/kg bw/d was associated with an effect on male fertility, the exact aetiology of which cannot be established based on current information.

 

The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day.

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 September 2014 and 13 April 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 422

Deviations:

yes

Remarks:

Some procedures were performed on Day 25 or 26 post coitum rather than post partum, as the group failed to achieve pregnancy.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for at
least six days during which time their health status was assessed. A total of ninety seven animals
(forty eight males and forty nine females(including one replacement animal)) were accepted into
the study. At the start of treatment the males weighed 296 to 346g, the females weighed 196 to
229g, and were approximately twelve weeks old.
Initially, all animals were housed in groups of three in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group. Following
evidence of successful mating, the males were returned to their original cages. Mated females
were housed individually during gestation and lactation in solid floor polypropylene cages with
stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during gestation and lactation.
Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
The diet, drinking water, bedding and environmental enrichment was considered not to contain
any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. There were no deviations from the target
range for temperature and transient deviations from the target range for relative humidity were
considered not to have affected the purpose or integrity of the study; see deviations from Study
Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study.
Results show the formulations to be stable for at least seventeen days in the dark at approximately 4 °C. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.
Samples of the test item formulation were taken and analyzed for concentration of 1,3- Propanediol, 2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with (chloromethyl) oxirane CAS RN: 30499-70-8 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 94% to 107% of the nominal concentration confirming the accuracy and suitability of the formulation procedure.

One female (37R) at 30mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen right eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC / UV) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

Test item
The test item described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot, 250 mg of test item was accurately weighed into a 2500 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 0.1 mg/L. Aliquots of this stock standard solution were used to prepare working standard solutions in water with a concentration of 0.001 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile this was then shaken to dissolve. Where necessary, sample solutions were further diluted with acetonitrile (intermediate dilutions) and then water (final dilutions) to achieve the working concentration.

Preparation of accuracy samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

Instrument set up
HPLC system: Agilent Technologies 1200 MSD, incorporating autosampler and work station
Mass selective detector
Source: Electrospray
Fragmentation energy: 90 100, 150 and 160 volts
Polarity: positive
Mode: single ion mode with 247.1, 269.2. 320.3, 571.2 amu
Gas temperature: 350 °C
Drying gas: 11.1 L/min
Nebuliser pressure: 40 psi
Capillary voltage: 1500 volts
Gain: 1
Column: Luna C18, 5 µ (150 x 2 mm id)
Column temperature: 40 °C
Gradient elution:
Eluent A: 0.1 % formic acid in LC-MS water
Eluent B: 0.1 % formic acid in acetonitrile

Time (mins) %A %B
0 80 20
5 0 100
10 0 100

Flow rate: 0.4 mL/min
Injection volume: 10 µL
Retention time: Approximately 5 to 7 minutes

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Results of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method and retention time was confirmed.

Linearity
The data was found to have a quadratic correlation within the calibration range of 0.005 to 0.0015 mg/L. The R2 fit of the calibration curve to the data was 0.996 and was considered to be acceptable.

Accuracy
The fortified samples of PEG 400 were found to have a recovery value of ± 10 % of the fortification.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 94 – 107 % and, thus, the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 17 days in the refrigerator (4 °C).
In conclusion, the results indicate the accurate use of the test item and PEG 400 as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable correlation to the fit of a quadratic equation. The analytical procedure was found to have acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.

Details on mating procedure:

On Day 15 (Day 16 for male 25 and female 37), animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

Duration of treatment / exposure:

Up to 56 days

Frequency of treatment:

Daily

Duration of test:

Up to 56 days

Remarks:

Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Maternal examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and approximately one hour after dosing. All observations were recorded.


Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.


Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:

Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.


Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

One female (37R) at 30 mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen right eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study. The replacement female was placed in the same cage as the original female. As the cage always contained the correct number of animals it was considered acceptable to continue with the food consumption for this cage and use it in the assessment of effects during the first week of the study although the initial food intake would have included a contribution from the excluded animal.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids


Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum and Day 25 post coitum for females at 300 mg/kg bw/day. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.


All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group:

Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

Tissues shown in bold were weighed from all remaining animals:
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

As all females were non-pregnant at 300 mg/kg bw/day, normal range data based on 90 Day Toxicity studies are also present for female (non-pregnant) animals.


Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:

Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes•, Liver, Thymus, Lungs (with bronchi) #, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina.

* = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 300 mg/kg bw/day dose group animals, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in below from the remaining control and 300 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. Female 37R was excluded from histopathological processing and investigation. In addition, sections of testes from all control and 300 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Testes, Uterus/Cervix, Vagina, Mammary gland

Detailed qualitative examination of the testes was undertaken, 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, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

As there were no indications of treatment-related changes, examination was not extended to include animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (Jeffrey Wilson at Propath GmbH, Muttenzerstrasse 30, 4133 Pratteln, Switzerland). A peer review of findings was conducted by the Test Facility.

Ovaries and uterine content:

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). Where possible the corpora lutea were also counted.

Fetal examinations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)


Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females, Day 25 post coitum for females receiving 300 mg/kg bw/day). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Statistics:

See below

Indices:

See below

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths on the study that were attributed to treatment.

One female (37R) at 30 mg/kg bw/day was killed for animal welfare considerations on Day 2 after showing a dark, swollen eye. This death was clearly unrelated to treatment and, in view of its the close proximity to the start of the study, this female was replaced and excluded from the assessment of toxicity for this study.

Clinical Observations
Clinical signs associated with treatment during the study were restricted to increased post-dosing salivation for all males and, to a lesser extent, the majority of females at 300 mg/kg bw/day. Increased post-dosing salivation is frequently observed when a slightly unpalatable or irritant test item is administered via the oral gavage route and is generally considered to reflect distaste of the dosing formulations rather than any adverse systemic effect of treatment.

At 100 mg/kg bw/day, one male showed incidences of pilo-erection on two separate occasions during the study, but in the absence of any similar findings at 300 mg/kg bw/day, this isolated finding was considered to be incidental and unrelated to treatment.

No clinical signs were apparent during the study for both sexes at 30 mg/kg bw/day or for females at 100 mg/kg bw/day.


Functional Observations
There were no abnormal observations apparent during behavioural assessments and there was no consistent pattern in intergroup differences for behavioural assessment scores that indicated any effect of treatment.

Functional Performance Tests
There were no changes in functional performance that were considered to be related to treatment.

For females at 30 mg/kg bw/day, lower mean values for the last 20% mobile activity attained statistical significance when compared with control but, in the absence of any similar effect at higher dosages this finding was considered to be incidental and unrelated to treatment.

Sensory Reactivity Assessments
Inter-group differences in sensory reactivity scores did not indicate any effect of treatment for either sex at 30, 100 or 300 mg/kg bw/day.


Body Weight
Body weight gain of males was considered to be unaffected by treatment throughout the study at 30, 100 and 300 mg/kg bw/day.

At 300 mg/kg bw/day, body weight gain of males was slightly lower than control during the final week of the study with differences attaining statistical significance. Group mean body weights are influenced by the laboratory investigations being performed during this period and, as body weight gains prior to this period, were essentially similar to control, these differences were considered to reflect normal biological variation rather than any treatment related effect of the Test Item.

Body weight gain of females was considered to be unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Body weight gain of females during pregnancy and lactation was considered to be unaffected by treatment at 30 and 100 mg/kg bw/day. Body weight gain during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.


Food Consumption
At 300 mg/kg bw/day, food consumption for males was lower than control during the first week of treatment; thereafter food consumption was essentially similar to control and was unaffected by treatment.

There was no obvious effect on food consumption for males at 30 and 100 mg/kg bw/day.

Food consumption of females was considered to be unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Food consumption of females during pregnancy and lactation was considered to be unaffected by treatment at 30 and 100 mg/kg bw/day. Food consumption during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.


Food Conversion Efficiency
Food conversion efficiency for both sexes during the pre-pairing phase and males during the post-mating phase of the study was considered to be unaffected by treatment at 30, 100 and 300 mg/kg bw/day.


Water Consumption
Visual assessment of water consumption during the study did not indicate any obvious effect of treatment on water intake for either sex at 30, 100 or 300 mg/kg bw/day.

Reproductive Performance
Mating and Fertility
There were no treatment-related effects on mating performance as assessed by pre-coital interval, with all treated animals mating within the first four days of pairing (i.e. at the first estrus opportunity); however at 300 mg/kg bw/day none of the matings resulted in a pregnancy.


Gestation Length
There was a tendency for gestation length to be longer than control for females receiving 30 or 100 mg/kg bw/day with differences from control attaining statistical significance: however all gestation lengths were within the normally expected range observed within this laboratory for this strain of rat.

Laboratory Investigations
When assessing the results of laboratory investigations for females at 300 mg/kg bw/day, it has to be considered that all these females were non-pregnant and were therefore in a different physiological state to the remaining females on the study.


Hematology
Intergroup differences for a number of haematology parameters for females at 30 and 100 mg/kg bw/day and both sexes at 300 mg/kg bw/day attained statistical significance when compared with control but none of these findings were supported by any histopathological change and, at the levels observed, these were considered not to indicate any adverse effect of treatment.

For males at 300 mg/kg bw/day, higher erythrocyte counts and haemoglobin levels attained statistical significance but all individual values for these treated animals were within the historical control range.

For females at 300 mg/kg bw/day, higher erythrocyte count, hemoglobin and hematocrit level and lower mean cell hemoglobin and mean cell volume attained statistical significance when compared with control. With the exception of one value for erythrocyte count and hemoglobin, values for these treated animals were within the historical control range, while for the control females, one value for erythrocyte count, mean cell hemoglobin and mean cell volume were outside this historical range. Statistically significantly lower mean cell volume was also observed for females at 30 and 100 mg/kg bw/day but all individual values were within the historical control range.

For females at all dosages, total leukocyte count and the number of neutrophils were statistically significantly lower than control; all values for these treated animals were within the historical control range, while one control value for total leukocyte count and two control values for neutrophils were outside this historical range. At 300 mg/kg bw/day, higher numbers of lymphocytes attained statistical significance when compared with control but only one value for these treated animals exceeded the historical control range.

For females at 100 and 300 mg/kg bw/day, lower platelet counts attained statistical significance compared to control; all individual values for these treated animals were within the historical control range but one control value exceeded this historical range.

For females at 30 mg/kg bw/day, there was a statistically significant increase in prothrombin time compared to control; there was no similar increase at higher dosages and all individual values for these treated animals were within the historical control range.

Blood Chemistry
Intergroup differences for a number of blood chemistry parameters for females at 30 and 100 mg/kg bw/day and both sexes at 300 mg/kg bw/day attained statistical significance when compared with control but none of these findings were supported by any histopathological change and, at the levels observed, these were considered not to indicate any adverse effect of treatment.

For males at 300 mg/kg bw/day, higher creatinine levels attained statistical significance compared to control; this was principally due to one treated animal with a particularly high recorded creatinine level, all other values were within the historical control range.

For females at all dosages, albumin/globulin ratio was statistically significantly higher than control but there was no consistent dosage relationship. Additionally, there were no accompanying statistically significant changes in the levels of total protein or albumin and all values for albumin/globulin ratio within the historical control range.

For females at 300 mg/kg bw/day, higher glucose, calcium and inorganic phosphorus levels attained statistical significance compared to control; two glucose values and inorganic phosphorus values exceeded the historical control, all other values were within this historical range.

For females at 300 mg/kg bw/day, lower alanine aminotransferase and alkaline phosphatase levels attained statistical significance compared to control; all values were within the historical control and a decrease in these parameters is unlikely to indicate an adverse effect of treatment.

Necropsy
Adults
Neither the type, incidence or distribution of macroscopic findings apparent at terminal necropsy indicated any obvious effect of treatment at 30, 100 or 300 mg/kg bw/day.
Organ Weights
At 300 mg/kg bw/day, there was a decrease in absolute and body weight relative liver weights for females compared with control, with differences attaining statistical significance. These differences are considered to reflect the difference in pregnancy state between the pregnant controls and the non-pregnant treated females. Body weight relative values are considered to be the best indicator of toxicological effect for this organ and all body weight values for these treated animals were within the historical range based on Ninety Day Toxicity studies where females are of similar age and the same pregnancy status.

At 300 mg/kg bw/day, there was an increase in absolute and body weight relative thymus weights for females compared with control, with differences attaining statistical significance. Again, these differences were probably influenced by the difference in pregnancy state between the pregnant controls and the non-pregnant treated females; all individual thymus weights were within the historical range based on Ninety Day Toxicity studies.

At 300 mg/kg bw/day, statistically significantly lower absolute and body weight-relative heart weights, compared to control, were apparent but all values for treated animals were within the historical control range.


Histopathology
Microscopic examination of tissues for both sexes at 300 mg/kg bw/day did not reveal any microscopic alterations indicative of toxicity. There was no evidence of histopathological changes that could give an explanation for the failure of females to achieved pregnancy at this dosage.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
As previously discussed, all females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it is not possible to assess litter responses at this dosage. All control females and females receiving 30 or 100 mg/kg bw/day achieved pregnancy, gave birth and successfully maintained a litter to Day 4 of lactation.


Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30 or 100 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.


Offspring Growth and Development
There was no adverse effect on offspring bodyweight and litter weights at Day 1 and subsequently body weight gain to 4 post partum at 30 or 100 mg/kg bw/day. At 100 mg/kg bw/day, offspring body weights were slightly higher than control on Day 1 of age, with differences for males attaining statistical significance. These differences were considered to reflect the slightly longer gestation period observed for parent females at this dosage rather than any treatment related effect on pre-natal/early post-natal growth.

Offspring performance during assessment of surface righting appeared to be unaffected by maternal treatment at 30 or 100 mg/kg bw/day.

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 30 or 100 mg/kg bw/day.

Necropsy
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal treatment at 30 or 100 mg/kg bw/day.

Remarks on result:

other: all females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it is not possible to assess litter responses at this dosage.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

other: offspring growth and development

Remarks on result:

other: All control females and females receiving 30 or 100 mg/kg bw/day achieved pregnancy, gave birth and successfully maintained a litter to Day 4 of lactation.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. There was a clear effect on reproduction at this dosage leading to no pregnant animals being available for assessment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 300 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period. Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Surviving adult males were terminated on Day 43 or Day 44, with the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results…….

There were no adult deaths during the study that were attributed to treatment. Clinical observations identified an increase

salivation post-dosing for all males and the majority of females at 300 mg/kg bw/day.

 

There were no treatment-related changes in the body weight, consumption, behavioural parameters, functional performance or sensory reactivity at 30, 100 or 300 mg/kg bw/day. Body weight gain of females was unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Body weight gain of females during pregnancy and lactation was unaffected by treatment at 30 and 100 mg/kg bw/day. Body weight gain during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.

 

Food consumption of females was unaffected by treatment during the two week pre-pairing period at 30, 100 and 300 mg/kg bw/day. Food consumption of females during pregnancy and lactation was unaffected by treatment at 30 and 100 mg/kg bw/day. Food intake during pregnancy and lactation could not be assessed at 300 mg/kg bw/day as no females at this dosage achieved pregnancy.

Pre-coital interval was unaffected by treatment study at 30, 100 and 300 mg/kg bw/day, but at 300 mg/kg bw/day none of the matings resulted in a pregnancy. There was a tendency towards longer gestation length for females at 30 and 100 mg/kg bw/day.

All females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it is not possible to assess litter responses at this dosage. All control females and females receiving 30 or 100 mg/kg bw/day achieved pregnancy, gave birth and successfully maintained a litter to Day 4 of lactation. 

 

Corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size, sex ratio and offspring survival to Day 4 of age was unaffected by maternal treatment at 30 or 100 mg/kg bw/day. There was no adverse effect on treatment on offspring bodyweight and body weight gain, litter weights, clinical signs, performance of surface righting or necropsy findings at 30 and 100 mg/kg bw/day.

 

There was no adverse effect of treatment on hematology or blood chemistry parameters for both sexes at 30, 100 or 300 mg/kg bw/day. No statistically significant differences were observed for organ weights that were considered to indicate an adverse effect of treatment. Macroscopic necropsy findings did not indicate any effect of treatment for either sex at 30, 100 or 300 mg/kg bw/day. Microscopic examination of tissues for both sexes at 300 mg/kg bw/day did not reveal any microscopic alterations indicative of toxicity or any evidence of histopathological change that could give an explanation for the failure of females to achieved pregnancy at this dosage.

 

Conclusion

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. There was a clear effect on reproduction at this dosage leading to no pregnant animals being available for assessment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st May 2018 to 24th September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Chemical name (IUPAC) : Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane
CAS No. : 30499-70-8
Physical appearance : Clear colourless liquid
Purity as per
Certificate of Analysis
: 58% C15H26O6 and 25% C12H22O5
Batch No. : AAF1078500
Manufactured date : 22.09.2016
Expiry Date : 07.09.2019
Storage Conditions : Ambient (+15°C to +25°C)
Physico-chemical
properties
: pH: 7 at 20 degrees C
Density: 1.12g/cm3 25degrees C

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain : Wistar Rats- HanTac: WH
Source : Vivo Bio Tech Ltd, Sy # 349/A,
Pregnapur-502311, Gajwel Mandal,
Medak District, Telangana, India

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item was administered orally by gavage using disposable plastic
syringe attached with a metal feeding cannula to rats of specific groups daily
from GD 5 to GD 19 of presumed gestation, at approximately the same time
each day (varying by ± 3 hours). Test item was administered at an equivolume
of 5 mL/kg body weight. The actual volume was calculated for individual
animals using the most recent body weight.
The animals in the vehicle control (G1) group were handled and administered
vehicle (PEG 400) in an identical manner to the treatment groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For dose formulation analysis, prepared formulations were sampled at the prior
to the initiation (08 June 2018) and a day before the termination (25 June 2018)
of batch 1 of rats.
The prepared formulations was sampled in duplicate sets wherein one set was
used for analysis and another was kept as back up set which was stored at room
temperature in the experimental room. For each set, one replicate sample was
drawn from top, middle and bottom layers for each dose formulation. In case of
control, one sample from middle layer was drawn. Dose formulations were sent
for formulation analysis to determine the concentration and homogeneity of the
test item using a validated analytical method (Study No.: G16353).
Formulations were considered acceptable if the mean result for all layers were
within ± 15% of the claimed concentration and the relative standard deviation
(RSD) of assay of top, middle and bottom layer (total 3 samples) was less than
or equal to 10%.
The unused back up samples was discarded as the results of first set of analysis
were within the acceptable limits.

Details on mating procedure:

vaginal smears and or vaginal plug was examined in the morning hours of the
subsequent day. If sperm was detected in a vaginal smear and / or if a vaginal
plug was observed in the morning, the female was considered to be mated. This
day was considered as Day 0 of gestation.
The females were cohabited in batches of required numbers. This procedure
was continued until there are sufficient numbers of Day 0 pregnant rats for the
study.
Day 0 pregnant rats obtained on each day was randomly distributed to different
groups by body weight stratification method using ProvantisTM software. After
grouping and ascertaining the group mean body weight, the rats were given
accession number as applicable to the group on each day of assignment.
Note: After confirming the Day 0 of gestation, females were housed
individually and the unselected females and the males were euthanized. The
males will be disposed or returned to the stock after mating procedure

Duration of treatment / exposure:

The test item was administered orally by gavage using disposable plastic
syringe attached with a metal feeding cannula to rats of specific groups daily
from GD 5 to GD 19 of presumed gestation, at approximately the same time
each day (varying by ± 3 hours). Test item was administered at an equivolume
of 5 mL/kg body weight. The actual volume was calculated for individual
animals using the most recent body weight.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels of 30 (G2), 90 (G3) and 180 (G4) mg/kg/day were selected for
this study based on the available literature in consultation with the Sponsor.
In addition to the test doses, vehicle control group was included. Animals in
the vehicle control group were handled in a manner similar to the treatment
groups except for test item administration.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes
Observations for clinical signs were performed twice a day -pre dose and post
dose (30 minutes to 1 hour post dose) during treatment days and once on nontreatment
days. On 30 June 2018 post dose observation was delayed and it was
carried out 2 hours after the dose.

BODY WEIGHT: Yes
All females included in the study were weighed on gestation days 0, 3, 5, 8, 11,
14, 17 and 20.
From this, intermittent body weight gain i.e., 0 - 3, 3 - 5, 5 - 8, 8 -11, 11- 14,
14 - 17, and 17 - 20 were calculated. Further the body weight gain for pretreatment
period (0 - 5), treatment period (5 - 20) and for entire gestation
period (0 - 20) were derived. Summary of body weight data was statistically
analyzed only for rats found pregnant at caesarean section.
The corrected body weight gain was calculated by subtracting the body weight
on day five of gestation from the corrected body weight. The corrected body
weight was obtained by subtracting the unopened uterine weight from terminal
body weight (body weight on Day 20 of gestation).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
About 200 g of food (food input) was provided on Day 0 of presumed
gestation. The food output was recorded and replenished to a known weight on
Days 3, 5, 8, 11, 14 and 17 and food output on Day 20 of presumed gestation
will be recorded. There was no spillage of food noticed during the entire study
period.
Food consumption was expressed as grams of food consumed daily per rat.
Intermittent food intake i.e., 0 - 3, 3 - 5, 5 - 8, 8 - 11, 11 - 14, 14 - 17 and 17 -
20 was calculated. Further food intake for pre-treatment period (0 - 5),
treatment period (5 - 20) and for entire gestation period (0 - 20) was derived.
Summary of food consumption data was statistically analyzed only for rats
found pregnant at caesarean section.

NECROPSY: Yes
Prior to caesarean section, random numbers were generated for coding to avoid
bias during caesarean section and subsequent fetal evaluations. The animal
code was written on the tail by striking out the permanent accession number.
All rats on gestation day 20 (at caesarean section), were subjected to postmortem
examination by sacrificing them under isoflurane anaesthesia. Gross necropsy, involved an external observation and examination of thoracic and
abdominal viscera including uterine contents.

OTHER:

Ovaries and uterine content:

Caesarean section: The gravid uterus along with the cervix was excised,
weighed and immediately examined.
The following maternal data were recorded.
• Pregnancy status
• Gravid Uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta
Uteri that appeared non-gravid were subjected to 10 % ammonium sulphide
staining to observe implantation sites (identified as pregnant animals) or to
confirm the non-pregnant status.

Fetal examinations:

The individual fetuses were removed sequentially. The fetuses were euthanised
using isoflurane anaesthesia.
The following litter data were recorded:
• Total number of fetuses
• Number of live fetuses
• Number of dead fetuses
• Individual fetal body weight (g)
• Fetus sex (external determination based on anogenital distance)
On GD 20, fetuses were delivered by hysterectomy and evaluated for external,
visceral and skeletal examinations.

Approximately one half of the fetuses from each litter were subjected to
visceral evaluation and the remaining half of the fetuses were subjected to
skeletal evaluation.
Fetal Visceral Evaluation: Approximately one half of the fetuses from each
litter was prepared for fresh tissue visceral organ evaluation. The fetuses
subjected for visceral evaluation was decapitated and the heads were stored in
70 % alcohol for sectioning using modified Wilsons Razor blade sectioning
technique.
Fetal Skeletal Evaluation: The remaining half of the fetuses of a litter were
prepared for skeletal evaluation by wet skinning followed by evisceration and
staining. Fetuses were fixed in 70 % ethyl alcohol, eviscerated and dehydrated
in 95 % ethyl alcohol; macerated in 1.5 % KOH and stained with saturated,
aqueous Alizarin red S in Mall’s solution. The excess stain was removed in
Mall’s solution and fetuses were cleared by passing through grades of glycerol
with thymol crystals.
Individual bottles containing fetal heads and fetuses subjected for skeletal
evaluation were labeled with study number, code number and fetal number
among total number of fetuses.
After evaluation, all the skeletal specimens were pooled per dam and preserved
in 100% glycerol with thymol crystals to prevent fungal growth. Fetal heads
subjected for sectioning were pooled dam wise and stored in formalin. The
bottles were labeled with study number, code number and number of
fetuses/head evaluated among total no of fetuses.
All recorded findings are presented in the report as per the standard reporting
format. The results of the study are discussed relative to the Test Facility
historical control data.

Statistics:

The data on maternal body weight, body weight change in interval, gravid
uterine weight, body weight change corrected to gravid uterine weight,
maternal food consumption were analyzed using Analysis of Variance
(ANOVA) after testing for homogeneity for intra group variance using
Levene’s test. Where intra group variances are heterogeneous, ANOVA was
performed after suitable transformation of data. Dunnett’s pairwise comparison
of the treated group means with the control group mean was performed, when
the group differences are found significant.
Fetal weight for male and female was analyzed using Analysis of Covariance
(ANCOVA) taking litter size as covariate for group.
Number of corpora lutea, number of implantations, early and late resorptions,
pre-implantation and post-implantation loss, external, visceral and skeletal
observations for variations were analyzed using Kruskal Wallis test for group
comparison. Mann-Whitney pairwise comparison of the treated groups with
the control group was performed, when the group differences were significant.
The incidence of dams with and without resorptions was tested using Cochran
Armitage trend test followed by Fisher’s exact test for group association.
Statistically significant differences (p<0.05) were designated as * throughout
the report.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The maternal parameters (mean gravid uterine weight, mean numbers of
corpora lutea, mean numbers of implantations, early and late resorptions, preand
post-implantation losses) were comparable between the vehicle control and
rats treated up to 180 mg/kg/day.

The litter parameters (mean fetal weight and number of live fetuses) were
comparable between the vehicle control group and rats treated up to
180 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: 180 mg/kg/day was without effect on maternal body weights, weight gain, food consumption and the maternal and litter parameters were comparable to vehicle control group. There were no gross pathological changes at any dose level.

Key result

Abnormalities:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

180 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 180 mg/kg/day was without effect on maternal animals and litter parameters were comparable to vehicle control group. There were no gross pathological changes at any dose level.

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In this study, prenatal developmental toxicity of Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane was
evaluated in Wistar rats following administration once daily by oral gavage at
0, 30, 90 and 180 mg/kg/day during gestation days 5 to 19. Reaction mass of 1-(2,3-epoxypropoxy)-2,2-bis ((2,3-epoxypropoxy)methyl) butane and 1-(2,3-epoxypropoxy)-2-((2,3-epoxypropoxy)methyl)-2-hydroxymethyl butane
up to 180 mg/kg/day was without effect on maternal body weights, weight
gain, food consumption and the maternal and litter parameters were
comparable to vehicle control group. There were no gross pathological changes
at any dose level. Gross evaluation of the placenta revealed no findings.
External, visceral and skeletal examination of fetuses revealed no signs of
teratogenicity.
Based on the above findings, the No- Observed- Adverse- Effect Level
(NOAEL) for maternal toxicity and fetal developmental toxicity is
180 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental

toxicity when administered daily by oral gavage during

gestation days (GD) 5 to 19 to presumed pregnant Wistar rats. The results of

this study helped to establish the No Observed Adverse Effect Level (NOAEL)

of the test item for maternal and developmental toxicity.

Ninety six presumed pregnant Wistar rats were assigned to four groups by

body weight stratification (Group 1 to Group 4) and each group (G1: control,

G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed

pregnant rats (gestation day 0). Day `0' of gestation for each individual female

rat in the study was considered as the day on which vaginal smear was found

positive for sperm.

1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-,

oligomeric reaction product with (chloromethyl)oxirane

in vehicle [Polyethylene Glycol 400 (PEG 400)] was

administered at 0, 30, 90 and 180 mg/kg/day. The control group received the

vehicle only. A constant dose volume of 5 mL/kg body weight was

administered to all groups.

The identity of the test item was provided by the study sponsor by a Test Item

Data Sheet. The stability and homogeneity of the test item in the vehicle was

established under Study No. G16353 at concentrations of 1 and 100 mg/mL

and the results of the study indicated that the test item was stable and

homogenous in the vehicle for 4 days when stored at room temperature. During

the conduct of the experiment, homogeneity and active ingredient analysis was

carried out from the dose formulation samples collected prior to initiation of

treatment and termination of treatment. The results of analysis of formulations

were within the acceptable limits.

All rats were observed for clinical signs, morbidity and mortality, body weight

changes and food consumption. Caesarean section was performed for all the

rats on GD 20 and dams were examined for gross pathological changes. The

uterus was removed by laparotomy, weighed and the contents were examined

for number of implantation sites, early and late resorptions and number of

fetuses. The number of corpora lutea in ovaries was counted. All the fetuses

were sexed, weighed and examined for external malformations. Approximately

half the number of fetuses from each dam was examined for visceral

malformations and the remaining half was evaluated for skeletal

malformations.

The main findings of the study are presented below:

• There were no mortalities, clinical signs or gross necropsy findings at

any of the doses tested.

• Mean body weights and gains, food consumption were comparable

across the tested doses.

• Mean values of maternal parameters comprising uterine weight and

number of corpora lutea, implantations, early resorptions, late

resorptions, pre-implantation loss and post implantation loss were

statistically comparable to the vehicle control group up to the highest

dose of 180 mg/kg/day.

• The litter parameters comprising total number of fetuses, number of live

fetuses, male and female fetal weights were statistically comparable to

vehicle control group up to the highest tested dose of 180 mg/kg/day.

• Fetal external, visceral and skeletal examination revealed no signs of

teratogenicity up to the highest tested dose of 180 mg/kg/day.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect

Level (NOAEL) for maternal toxicity and fetal developmental toxicity is

180 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

180 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study carried out to OECD guideline 414

Additional information

An OECD 422 Combined Repeat Dose Toxicity Screening Study was conducted to investigate the potential for adverse effects on systemic toxicity, reproductive toxicity and offspring development. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. However there was a clear effect on reproduction at this dose level, leading to no pregnant animals or offspring. All females at 300 mg/kg bw/day failed to achieve pregnancy and therefore it was not possible to assess litter responses at this dosage. There were no effects on development observed in the mid or low dose groups.

An OECD 414 Prenatal Development Toxicity Study was conducted to further investigate the potential for adverse effects of treatment on embryo-fetal development. All parameters were determined as comparable to control. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity and fetal development was 180 mg/kg bw/d, the highest dose used on study.

Justification for classification or non-classification

In an OECD 422 study, a clear effect on reproduction at a dose level of 300 mg/kg bw/d was observed leading to no pregnant animals being available for assessment. The No Observed Adverse Effect Level (NOAEL) for reproduction and the survival, growth and development of the offspring therefore was considered to be 100 mg/kg bw/day. Further investigatory testing suggests an apparent effect on male fertility.

In an OECD 414 study, all parameters tested were comparable to control upto the highest dose level of 180 mg/kg/d. There were no gross pathalogical changes at dose levels 0 -180 mg/kg/d.

As a result of the findings from the OECD 422 Combined Repeat Dose and Reproductive Development Screening study, the substance is classified as a Category 1B reproductive toxicant, in accordance with UN-GHS and CLP Criteria.