Bis(2-hydroxyethyl) sulphone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 19 May 2016 and 31 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

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 on Day 15 of the study, 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 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43 or 44 of the study, followed by the termination of all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and 1000 mg/kg bw/day dose groups was performed.

Results

Adult Responses:

Mortality: There were no unscheduled deaths during the study.

Clinical Observations: Throughout the study, there were no clinical signs considered to be related to treatment with the test item.

Behavioral Assessment: There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests: There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments: Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Body Weight: There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the dose administration period.

Food Consumption: There was no adverse effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the treatment period.

Water Consumption: Visual inspection of water bottles did not indicate any overt differences for the animals given the test item in comparison with controls.

Reproductive Performance:

Mating: There was no effect of treatment on mating performance. All animals mated within four days after pairing.

Fertility: There were no treatment-related effects in conception rates for test item-treated animals in relation to controls. All females were pregnant.

Gestation Lengths: There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability: There was no detrimental effect of treatment with the test item on corpora lutea count, preimplantation loss, number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development: There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs up to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Laboratory Investigations:

Hematology: No toxicologically significant effects were detected in animals of either sex at any dose level.

Blood Chemistry: No toxicologically significant effects were detected in animals of either sex at any dose level.

Pathology

Necropsy: Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect of treatment up to a dose level of 1000 mg/kg

bw/day.

Organ Weights: No toxicologically significant effects were detected in animals of either sex at any dose level.

Histopathology: Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

Conclusion

The oral (gavage) administration of BHES to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study. Additionally, The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

See effects on fertility section.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

An oral (gavage) combined repeat dose toxicity study with reproductive/developmental toxicity screening test in the rat (OECD 422) was conducted on the test substance.

The test material was administered daily by gavage to three groups each of twelve male and twelve female Wistar Han HsdRccHan:WIST strain rats, for up to 6 weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg/day.

No treatment-related effects were observed for the reproductive performance or litter response paramaters examined. Therefore a 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Based on the results of the OECD 422 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level tested. The substance is therefore not classified for reproductive toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

No affect to outcome of study, see section 'Overall Remarks'

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-hydroxyethyl) sulphone

EC Number:

219-948-3

EC Name:

Bis(2-hydroxyethyl) sulphone

Cas Number:

2580-77-0

Molecular formula:

C4H10O4S

IUPAC Name:

2-(2-hydroxyethanesulfonyl)ethan-1-ol

Test material form:

solid

Specific details on test material used for the study:

Identification : BHES
Physical State/Appearance : White solid block; the Sponsor confirmed that the test
item can be heated up to 100 °C
Chemical Name : Ethanon, 2,2'-sulfonylbis-
Chemical Formula : C4H10O4S
Purity : 99.64%
Batch Number : 160226
Date Received : 08 March 2016
Storage Conditions : Room temperature, in the dark
Expiry Date : 01 March 2017
No correction for purity was made.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of ninety six animals
(forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 318 to 372g, the females weighed 188 to 229g, and were
approximately twelve weeks old.

Animal Care and Husbandry:
Initially, all animals were housed in groups of four 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, Envigo RMS (UK) Limited, 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 Envigo Research Limited, 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%. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the
study are believed to be of value in predicting the likely toxicity of the test item to man.
The test item was administered once daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Distilled water.
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.

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water.
The dose levels were chosen in consultation with the Sponsor based on the results from previous toxicity work including a 14 Day dose range-finding study in the rat. In the range-finding study, administration of the test item to animals of either sex at dose levels of up to 1000 mg/kg bw/day was well tolerated. Based on these results, a high dose level of 1000 mg/kg bw/day was considered to be suitable for investigation in the present study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromoatography profile consisting of a profile of multiple peaks.
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the
formulations to be stable for at least twelve days when stored at approximately 4 °C, in the dark. Formulations were therefore prepared approximately weekly,
aliquoted and stored as above before use.
Samples of test item formulation were taken on three occasions and analyzed for concentration of BHES at Envigo Research Limited, Shardlow, UK, Analytical Services.
The results indicate that the prepared formulations were within 95% to 103% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met.

Duration of treatment / exposure:

approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control treatment group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low treatment group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate treatment group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High treatment group

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

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.

Chronological Sequence of 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, 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, five selected males per dose group were evaluated for functional/sensory responses to various stimuli (during Week 6).
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.
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 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. At Day 5
post partum, all females and surviving offspring were killed and examined macroscopically.

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 one hour after dosing (except for females during parturition where applicable). All observations were recorded.

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.
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 during gestation and lactation.

FOOD EFFICIENCY:
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 during gestation and lactation.

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

REPRODUCTIVE PERFORMANCE
1) 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. Female 65 from the 300 mg/kg bw/day dose group showed evidence of uncertain mating (copulation plugs observed in the cage only
but no sperm detected in vaginal smear) on Day 2 after pairing and was kept with the male until the end of the pairing period. Subsequently, this female produced a litter and it was
assumed to have mated on Day 2 after pairing. Mated females were housed individually during the period of gestation and lactation.

2) 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

3) 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)

4) PHYSICAL DEVELOPMENT
All live offspring were assessed for surface righting reflex on Day 1 post partum.


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

OTHER:
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.

BEHAVIOURAL ASSESSMENT:
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).

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
Finger approach
Startle reflex

Sacrifice and pathology:

NECROPSY
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of
suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent on Day 5
post partum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. 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. Tissues indicated by an * were
weighed from all remaining animals:
Adrenals *Pituitary (post-fixation)
Brain *Prostate and Seminal Vesicles
*Epididymides Spleen
Heart *Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
*Ovaries *Uterus (weighed with Cervix)

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 indicated by an * 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) *Testes ♦
Jejunum Thyroid/Parathyroid
Kidneys Trachea
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 got processing. The tissues from five selected control and 1000 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 marked with * from the remaining control and 1000 mg/kg bw/day animals as well any gross lesions from the remaining animals were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
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.

Statistics:

Data were processed to give summary incidence or group mean values and standard deviations where appropriate.

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 paired) 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.
Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Where considered appropriate, quantitative data was subjected to statistical analysis to detect
the significance of intergroup differences from control; statistical significance was achieved
at a level of p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations related to treatment with the test item at any dose level.
One male receiving the test item at a dose level of 300 mg/kg bw/day showed clinical signs of piloerection after dosing on Day 40 relative to the start of treatment. None of the
remaining animals on the study showed any clinical signs and this observation was deemed unlikely to be related to treatment with the test item.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the dosing period, there was no effect of treatment with the test item on body weight development in animals of either sex.
At all dose levels, weekly group mean body weight gains in males were generally comparable with controls throughout the dose administration period. Females from the test item-treated dose also showed similar body weight gains to controls before pairing. During gestation and lactation phases of the study, body gains for females receiving the test item up to a dose level of 1000 mg/kg bw/day were generally comparable with controls. Any minor intergroup differences were neither dose-dependent nor achieved statistical significance and were deemed unlikely to be related to administration of the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Throughout the dosing period (including gestation and lactation phases for the females), there was no effect of treatment with the test item on dietary intake in animals of either sex.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item on food conversion efficiency in animals of either sex receiving the test item. Any small differences were deemed to be reflective of
minor intergroup differences in body weight gain and/or food consumption.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not indicate any overt differences for the animals given the test item in relation to controls.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.
At all dose levels, females treated with the test item showed statistically significantly lower mean corpuscular volume in relation to controls (p<0.05). There was no dose-relationship and with the exception of one female from the 300 mg/kg bw/day dose group, individual values from the remaining females were within the historical control data ranges. Group mean values in the corresponding males were similar to controls and this observation was considered unlikely to be of any toxicological significance.
The remaining haematology parameters in animals of either sex were generally similar to controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry:
No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.
At 300 or 1000 mg/kg bw/day, females receiving the test item showed statistically significantly lower plasma levels of sodium when compared with controls (p<0.05). There was no clear dose-relationship and all individual values remained within the historical control data ranges. Group mean values in the corresponding males were similar to controls and, in the absence of any effect on associated parameters or any histopathology correlates, this observation was deemed unlikely to be of any toxicological relevance.
Group mean total protein concentration in males from the 100 mg/kg bw/day dose group was statistically significantly higher than controls (p<0.05). The corresponding values in the remaining male and female dose groups were similar to controls and this finding was considered to be incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no intergroup differences considered to be related to treatment with the test item.
Motor activity evaluation during the final week of dosing revealed slightly but statistically significantly higher overall activity for females given the test item at 300 mg/kg bw/day in relation to controls (p<0.05). A dose-relationship was not evident and, in the absence of a similar effect in the remaining animals or any clinical signs of neurotoxicity, this observation was deemed unlikely to be treatment-related.
Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with controls, absolute and body weight-related group mean thyroid/parathyroid weights in females from all dose groups were statistically significantly higher than controls (p<0.05) with females treated with 300 or 1000 mg/kg bw/day showing statistically significantly lower absolute and body weight-related thymus weights (p<0.05). There was no dose-dependence and all individual values were within the historical control data ranges. Females receiving 1000 mg/kg bw/day also showed statistically significantly lower absolute and body weight-related ovaries weights in relation to controls (p<0.05) but without any dose-relationship and with most individual values remaining within the historical control data ranges. In the absence of any associated histopathology observations, these findings were deemed unlikely to be of any toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were noted in any animals which could be related to treatment with the test item.
At necropsy, a number of animals of either sex receiving the test item exhibited red discoloration of the lungs. Such observations are common in this type of study and, in the absence of any associated histopathology findings, were considered unlikely to be treatment related.
One female treated with 300 mg/kg bw/day was observed with a mass (fibrous appearing) around the mammary gland region. This correlated with the microscopic finding of adenocarcinoma of the mammary gland, but in the absence of similar findings in the high dose group, this observation was considered to be unrelated to the test item administration

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Minor changes were noted in several tissues but these were not considered to be related to administration of the test item.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities
in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Mating:
There was no effect of treatment on mating performance with all animals mating within four days after pairing.

Fertility:
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level. All females were pregnant and successfully reared their young to Day 5 post partum.

Gestation Length:
Gestation lengths were between 22 and 23.5 days and the distribution of gestation lengths for test item-treated females was generally comparable with controls.

Litter Responses:
Offspring Litter Size, Sex Ratio and Viability:
There was no detrimental effect of treatment with the test item on corpora lutea count, pre-implantation loss, number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development:
There was no detrimental effect of treatment with the test idicated by offspring body weight or body weight gan and litter weights, surface righting ability on Day 1 or clinical signs up to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Effect levels

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At dose levels of 100, 300 or 1000 mg/kg bw/day the substance was well tolerated and no treatment related adverse effects were reported.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of BHES to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. Based on the available
results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.
Additionally, the 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

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 100, 300 or 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

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 on Day 15 of the study, 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 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43 or 44 of the study, followed by the termination of all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and 1000 mg/kg bw/day dose groups was performed.

Results

Mortality: There were no unscheduled deaths during the study.

Clinical Observations: Throughout the study, there were no clinical signs considered to be related to treatment with the test item.

Behavioral Assessment: There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests: There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments: Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Body Weight: There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the dose administration period.

Food Consumption: There was no adverse effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the treatment period.

Water Consumption: Visual inspection of water bottles did not indicate any overt differences for the animals given the test item in comparison with controls.

Reproductive Performance:

Mating: There was no effect of treatment on mating performance. All animals mated within four days after pairing.

Fertility: There were no treatment-related effects in conception rates for test item-treated animals in relation to controls. All females were pregnant.

Gestation Lengths: There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability:

There was no detrimental effect of treatment with the test item on corpora lutea count, pre-implantation loss, number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development:

There was no detrimental effect of treatment with the test idicated by offspring body weight or body weight gan and litter weights, surface righting ability on Day 1 or clinical signs up to Day 5 of age at 100, 300 or 1000 mg/kg bw/day.

Laboratory Investigations:

Hematology: No toxicologically significant effects were detected in animals of either sex at any dose level.

Blood Chemistry: No toxicologically significant effects were detected in animals of either sex at any dose level.

Pathology:

Necropsy: Neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect of treatment up to a dose level of 1000 mg/kg bw/day.

Organ Weights: No toxicologically significant effects were detected in animals of either sex at any dose level.

Histopathology: Histopathological examination of the selected tissues from the 1000 mg/kg bw/day animals of either sex did not reveal any treatment-related abnormalities.

Conclusion

The oral (gavage) administration of BHES to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. Based on the available results, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Additionally, the 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.