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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

701-135-4

Cas Number:

30499-70-8

Molecular formula:

C15H26O6;C15H22O5

IUPAC Name:

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

Test material form:

other: Liquid

Specific details on test material used for the study:

Identification : 1,3-Propanediol, 2-ethyl-2-(hydroxymethyl)-, oligomeric reaction product with (chloromethyl)oxirane (TK30174)
CAS RN: 30499-70-8
Physical State/Appearance : Clear colorless liquid
Purity : 58% C15H26O6 and 25% C12H22O5
Batch Number : AAB1584600
Date Received : 30 June 2014
Storage Conditions : Ambient 10 to 30 °C in the dark
Expiry Date : 31 October 2015

Test animals

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral 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.


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.

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100, 300 mg/kg bw/day
Basis:
nominal in diet

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

Examinations

Observations and examinations performed and frequency:

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

Litter Data
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.


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

Evaluation of Data
Treatment of Data
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

Data shown in the appendices are frequently rounded values for presentation purposes. Group mean values are generally calculated using non-rounded values therefore is it not always possible to calculate the exact group values from the individual values presented in the appendices.

As previously discussed, female 37R was a non treatment-related mortality. Data for this animal are not normally presented unless otherwise indicated but these data are retained with the raw data for the study.

For body weights and food consumptions during gestation, group mean values were calculated using data from females which were observed to give birth to offspring. At 300 mg/kg bw/day, no females achieved pregnancy and data has been presented based on Days post coitum, the differing pregnancy status of these females must be considered when evaluating the presented data.

For body weights and food consumptions during lactation, group mean values were calculated using data from females with live young at Day 5 of lactation.

Data for behavioral assessments, hematology, blood chemistry and organ weights were intended to be presented for females on Day 4 or 5 post partum, as appropriate. At 300 mg/kg bw/day, no females achieved pregnancy and data has been presented based on Day 25 or 26 post coitum as appropriate, the differing pregnancy status of these females must be considered when evaluating the presented data.
Reproductive Indices
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:

Mating Index (%) = (number of animals mated / number of animals) x 100

Pregnancy Index (%) = (number of pregnant females / number of animals mated) x 100


Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:

Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) x 100

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = ((number of corpora lutea – number of implantation sites) / number of corpora lutea) x 100

Post–implantation loss (%) = ((number of implantation sites – total number of offspring born) / number of implantation sites) x 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live birth index (%) = (number of offspring alive on Day 1 / number of offspring born) x 100

Viability Index (%) = (number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

Number of male offspring / total number of offspring) x 100

Sacrifice and pathology:

Pathology
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 and offspring, 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.

Other examinations:

None specified

Statistics:

See below

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See results

Mortality:

mortality observed, treatment-related

Description (incidence):

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

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Adult Reponses
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.


Litter Responses
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.


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.


Pathology
Necropsy
Offspring

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.

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.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day. 

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group after at least 14 days of consecutive days of dosing, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partumor Day 25post coitumif lactating females were unavailable. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

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…….

Adult Responses

Mortality

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

 

Clinical Observations

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

 

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters at 30, 100 or 300 mg/kg bw/day.

 

 

Functional Performance Tests

There were no treatment-related changes in functional performance at 30, 100 or 300 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity scores at 30, 100 or 300 mg/kg bw/day.

 

Body Weight

Body weight gain of males was unaffected by treatment throughout the study at 30, 100 and 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

Food consumption of males was unaffected by treatment throughout the study at 30, 100 and 300 mg/kg bw/day.

 

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.

 

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 unaffected by treatment at 30, 100 and 300 mg/kg bw/day.

 

Water Consumption

Water consumption for both sexes was unaffected by treatment throughout the study at 30, 100 and 300 mg/kg bw/day.

 

Reproductive Performance

Mating and Fertility

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.

 

Gestation Lengths

There was a tendency towards longer gestation length for females at 30 and 100 mg/kg bw/day.

 

Litter Responses

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

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.

 

Offspring Growth and Development

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.

 

Laboratory Investigations

Hematology

There was no adverse effect of treatment on hematology parameters for both sexes at 30, 100 or 300 mg/kg bw/day.

 

Blood Chemistry

There was no adverse effect of treatment on blood chemistry parameters for both sexes at 30, 100 or 300 mg/kg bw/day.

 

Pathology

Necropsy

Macroscopic necropsy findings did not indicate any effect of treatment for either sex at 30, 100 or 300 mg/kg bw/day.

 

Organ Weights

There were no differences observed for organ weights that were considered to indicate an adverse effect of treatment.

 

Histopathology

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.