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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2012 and 06 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl hydrogen maleate
EC Number:
213-116-3
EC Name:
Butyl hydrogen maleate
Cas Number:
925-21-3
Molecular formula:
C8H12O4
IUPAC Name:
(2Z)-4-butoxy-4-oxobut-2-enoic acid
Test material form:
other: liquid
Details on test material:
Description, Identification and Storage Conditions
Sponsor's identification : Butyl Hydrogen Maleate
Description : Pale yellow liquid
Chemical name : Butyl hydrogen maleate
Purity : >95%
Lot number : 120224
Label : ST1795KS lot 120224
Date received : 20 March 2012
Storage conditions : Room temperature in the dark
Expiry date : 30 March 2013

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:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: ~12 weeks
- Weight at study initiation:
males: 299-326g
females: 186-223g
- Fasting period before study: No
- Housing: 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.
In a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility
- Diet: ad libitum - A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used.
- Water: ad libitum - Mains drinking water was supplied from polycarbonate bottles attached to the cage
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: The animals were acclimatised for seven days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ±15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 continuous light 12 dark

IN-LIFE DATES: The in-life phase of the study was conducted between 15 August 2012 (first day of treatment) and 10 October 2012 (final necropsy).

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. The results of the study are believed to be of value in predicting the likely toxicity of the test item to man and to screen for potential adverse effects on reproduction.
Vehicle:
arachis oil
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 Arachis oil BP. Prior to preparation, the test item was
filtered and the presence/absence of a precipitate was recorded. The filtrate was used for formulation and analysis. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least thirteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.
Samples of the test item formulation were taken and analysed for concentration of Butyl Hydrogen Maleate at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 25. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD OF ANALYSIS
Summary
The concentration of Butyl Hydrogen Maleate in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
Samples
The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.1 mg/mL.
Standards
Standard solutions of test item were prepared in methanol at a nominal concentration of 0.1 mg/mL.
Procedure
The standard and sample solutions were analysed by HPLC using the following conditions:

HPLC...................................... : ................. Agilent Technologies 1200, incorporating autosampler and workstation
Column................................. : ................. Sunfire C18 5μ (150 x 4.6 mm id)
Mobile phase........................ : ................. Methanol: 0.1% orthophosphoric acid (60:40 v/v)
Flow-rate.............................. : ................. 1 mL/min
UV detector wavelength..... : ................. 220 nm
Injection volume.................. : .................25 μL
Retention time...................... : ................ ~ 3.6 mins

Homogeneity Determinations
The test item formulations were assessed visually.
Stability Determinations
The test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for thirteen days.
Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.
Duration of treatment / exposure:
The test item was administered by gavage for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for
females).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control group
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Low treatment group
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Intermediate treatment group
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Remarks:
High treatment group
No. of animals per sex per dose:
12 males, 12 females per treatment group
Control animals:
yes, concurrent vehicle
Details on study design:
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/behavioural 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 (during the final week of treatment), 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.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was
also killed and examined macroscopically.
Positive control:
no positive control was used.

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends (except for females during parturition where applicable). 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/behavioural 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 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 colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
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 Behavioural 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, 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
Vocalisation
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 mating was evident. 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 for females during gestation and lactation.

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

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 oestrus 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.
Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours 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
Haematological 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.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte 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
Sacrifice and pathology:
Pathology
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
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).
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, Brain, Spleen, Heart, Kidneys, Thymus, Liver and Thyroid (weighed post-fixation with Parathyroid).
The following tissues were weighed from all remaining animals:
Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix) and Pituitary (post fixation)

Histopathology
Samples of the following tissues from five selected males and five selected females from each dose group were removed and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides♦
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and Gross lesions lumbar)
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes♦
Lungs (with bronchi) #
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cervix
Muscle (skeletal)
Vagina
_________________________________
* = eyes fixed in Davidson’s fluid
♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were despatched to the histology processing Test Site (Harlan Laboratories Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland) for processing. The tissues from five selected control and 175 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 175 mg/kg bw/day animals were also processed. In addition, sections of testes and epididymides from all control and 175 mg/kg bw/day males were also stained
with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related changes in the stomach, liver, kidneys and thyroid gland, examination was subsequently extended to include similarly prepared sections of of these tissues from five animals per sex from the low and intermediate groups.
Microscopic examination was conducted by the Study Pathologist at AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland.
The histology processing and histopathology examination phases of the study were performed in accordance with the Swiss Ordinance relating to Good Laboratory Practice, adopted May 18 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26 1997 by decision of the OECD Council [C(97)186/Final].
Other examinations:
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 for 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

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) x100

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 on Days 1 and 4 post partum, using the following formula:

(Number of male offspring/Total number of offspring) x 100
Statistics:
Please see details below in "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Increased salivation and noisy respiration were recorded for animals of either sex treated with 175 mg/kg bw/day, and also some instances for males treated with 100 mg/kg bw/day.

One male treated with 100 mg/kg bw/day showed abnormal gait on Day 29. The animal was subsequently terminated. This finding was attributable to a physical injury and was considered to be unrelated to treatment.

One female treated with 30 mg/kg bw/day was recorded with a mass from Day 33 onwards. This was an incidental finding, and as such, was considered to be unrelated to test item toxicity.

PLEASE REFER TO THE ATTACHED TABLE: "SUMMARY INCIDENCE OF DAILY CLINICAL OBSERVATIONS"
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths considered attributable to test item toxicity.

One male treated with 100 mg/kg bw/day was killed in extremis on Day 29 due to a physical injury. There were no further unscheduled deaths.

Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effects on body weight change were detected for males during the treatment period of for females during the pre-pairing, gestation or lactation phases of the study.

Males treated with 175 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 4 and a statistically significant reduction in body weight gain during Week 6. The significance achieved in each case was minimal (p<0.05) and the overall body weight gains during the treatment period for males from this group were essentially similar to controls. As such, these statistically significant differences were considered to have arisen incidentally and were considered not to represent an adverse effect of treatment.

PLEASE REFER TO THE ATTACHED TABLES: "GROUP MEAN BODYWEIGHT VALUES" and "GROUP MEAN BODYWEIGHT GAINS"
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effects on dietary intake were detected for males during the treatment period or for females during the pre-pairing, gestation or lactation phases of the study.
A statistically significant reduction in dietary intake was noted for females treated with 100 mg/kg bw/day during the final week of gestation. The significance achieved was minimal (p<0.05) and in the absence of a dose-related response, this finding was considered to have arisen incidentally.
No adverse effects on food conversion efficiency (the ratio of body weight gain to dietary intake) were detected for treated animals when compared to controls.

PLEASE REFER TO THE ATATCHED TABLE: "GROUP MEAN FOOD CONSUMPTION"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any significant intergroup differences.
An increase in water intake was noted in high and intermediate dose males for five days. This was not reported during the remainder of the treatment period, therefore not considered toxicologically significant.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant differences were detected in the haematological parameters investigated.

Males treated with 175 and 100 mg/kg bw/day showed a statistically significant reduction in mean cell haemoglobin concentration when compared to controls. All individual values were within the normally expected ranges, and a convincing dose-related response was not observed. In the absence of any change in associated haematological parameters, these reductions were considered to have arisen incidentally and were considered to be unrelated to treatment with the test item.

PLEASE REFER TO THE ATTACHED TABLE: "GROUP MEAN HAEMATOLOGICAL VALUES"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in bile acids was noted for males treated with 175 mg/kg bw/day when compared to controls (p<0.01), with all individual values outside of the normally expected ranges.

There were no further blood chemical changes considered to be of toxicological importance.

Albumin/globulin ratio was higher than controls for all male treatment groups. Although the majority of individual values were outside of the normally expected ranges, a corresponding change in total protein or albumin levels was not observed and a dose- related response was not observed. As such, these minimal increases (p<0.05) were considered to have a risen incidentally and were unrelated to test item toxicity.

Total protein levels were reduced for males treated with 30 mg/kg bw/day when compared to controls. In the absence of a dose-related response, this minimal difference was considered to have arisen incidentally and did not represent a true effect of treatment.

Males treated with 30 mg/kg bw/day showed a statistically significant increase in chloride levels when compared to controls. All individual values were within the normally expected ranges and a dose-related response was not evident. As such, the significance achieved (p<0.01) was considered to be unrelated to test item toxicity.

Females treated with 100 mg/kg bw/day showed a statistically significant increase in albumin levels when compared to controls with two values outside of the normally expected ranges. In the absence of a dose-related response and corresponding changes in total protein or albumin/globulin ratio detected in this dose group, this increase was considered to be unrelated to treatment with the test item.

Creatinine levels were lower for females treated with 175 mg/kg bw/day when compared to controls. Although one lower than expected value was seen in this group, there were two higher than expected controls values which were considered to contribute to the statistically significant reduction detected. As such, this was not considered to represent a true effect of treatment.

PLEASE REFER TO THE ATTACHED TABLE: "GROUP MEAN BLOOD CHEMICAL VALUES"
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased hepatic metabolism of thyroid hormones (T3/T4) due to hepatoceullular hypertrophy. The finding was deemed to represent a secondary effect and not to be adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects were detected for treated animals when compared to controls.

Any inter and intra group differences in scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

Functional Performance Tests
No treatment-related effects were detected for treated animals when compared to controls.

Statistical analysis of the data did not reveal any significant intergroup differences.

Sensory Reactivity Assessments
No treatment-related effects were detected in sensory reactivity scores.

Any inter and intra group differences were considered to be a result of normal variation for rats of the strain and age employed, and were of no toxicological importance.

PLEASE REFER TO THE ATTACHED TABLES: "GROUP MEAN FUNCTIONAL TEST VALUES" and "GROUP MEAN SENSORY REACTIVITY ASSESSMENTS_SCORES"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated at all dose levels showed an increase in liver weights, both absolute and relative to terminal bodyweights, with statistical significance achieved for males treated with 175 and 100 mg/kg bw/day (p<0.01) and for females treated at all treatment levels (p<0.05) when compared to controls.

Animals of either sex treated with 175 and 100 mg/kg bw/day, and females treated with
30 mg/kg bw/day showed a statistically significant increase in kidney weights, both absolute and relative to terminal body weights, when compared to controls (p<0.01).

Males treated with 175 mg/kg bw/day showed a statistically significant increase in thyroid weights both absolute and relative to terminal body weights when compared to controls (p<0.01).

There were no further changes considered to be related to treatment.

Females treated with 30 mg/kg bw/day showed a statistically significant increase in spleen weights, both absolute and relative to terminal body weights. The significance achieved was minimal and the increase was considered to be attributable to one higher than expected value. In the absence of a dose-related response, this increase was considered to have arisen incidentally and unrelated to test item toxicity.

PLEASE REFER TO THE ATTACHED TABLE: "GROUP MEAN ORGAN WEIGHTS WITH CORRESPONDING RELATIVE ORGAN WEIGHTS"
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Adults

There were no treatment-related macroscopic abnormalities detected.

Small prostate and seminal vesicles were observed for one control male. A mass was detected on the mammary gland for one female treated with 30 mg/kg bw/day. The animal which was killed in extremis on Day 29 showed a fore paw disconnected from the limb. These were all incidental findings considered to be unrelated to test item toxicity.

PLEASE REFER TO THE ATTACHED TABLE: SUMMARY INCIDENCE OF NECROPSY FINDINGS"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
STOMACH:
Forestomach hyperkeratosis and squamous cell hypertrophy (i.e. simple increase of mucosal thickness) were recorded for animals of either sex treated with 175 or 100 mg/kg bw/day. Focal squamous cell hyperplasia of forestomach was recorded for animals of either sex treated with 175 mg/kg bw/day.

PLEASE REFER TO THE FOLLOWING TABLE IN "ANY OTHER INFORMATION ON RESULTS": "Incidence and Mean Severity Grade of Main Findings in Stomach (terminal kill)"

LIVER:
Diffuse hepatocellular hypertrophy was recorded for animals of either sex treated at all dose levels. Among animals showing hepatocellular hypertrophy, single cell death of the hepatocyte was recorded in two males treated with 175 mg/kg bw/day.

PLEASE REFER TO THE FOLLOWING TABLE IN"ANY OTHER INFORMATION ON RESULTS": Incidence and Mean Severity Grade of Main Findings in Liver (terminal kill)

THYROID:
Increased incidence and/or severity of follicular cell hypertrophy were recorded for females treated at all dose levels and for males treated with 175 mg/kg bw/day.

PLEASE REFER TO THE FOLLOWING TABLE IN ANY OTHER INFORMATION ON RESULTS: "Incidence and Mean Severity Grade of Main Findings in Thyroid Gland (terminal kill)"

KIDNEYS:
Proximal tubular hypertrophy was recorded for animals of either sex treated with 175 and 100 mg/kg bw/day, and a dose-related response was evident.

In addition, increased incidence and severity of tubular degeneration and regeneration, tubular dilatation, casts, and increased incidence or severity of interstitial mononuclear cell infiltration were recorded for animals of either sex treated with 175 mg/kg bw/day. Pelvic urothelial cell hypertrophy was recorded in one female treated with 175 mg/kg bw/day, which was deemed to be a secondary change following the lesions described above.

PLEASE REFER TO THE FOLLOWING TABLE IN "ANY OTHER INFORMATION ON RESULTS": "Incidence and Mean Severity Grade of Main Findings in Kidneys (terminal kill)"

The stages of spermatogenesis were checked on completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control animals and the high dose animals.

As a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Sertoli cell vacuolation (bilateral) and vasculitis (unilateral) were recorded at a minimal severity in one control male which did not produce a pregnancy in its female partner. Such minimal lesions are often recorded in the testis of males not having problems in fertilisation, and were unlikely to be related to the absence of pregnancy in the female partner.

There were no obvious changes detected in the reproductive organs for the remaining animals which mated but did not achieve a pregnancy.

The incidence of muscle fibre degeneration seemed to be slightly higher in the high dose males (three males) compared to the control male (one male). However, such lesion at minimal severity (focal/sporadic) are often recorded in animals of this strain and age, and therefore, this was considered to be spontaneous and within the range of normal background lesion.

Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mammary gland adenocarcinoma was recorded in one female treated with 30 mg/kg bw/day. This is occasionally recorded in females of this strain and age, and abnormal hyperplastic mammary gland lesions were not observed in animals treated with 175 and 100 mg/kg bw/day. Therefore, one case recorded in this study was considered to be a spontaneous lesion.

The remainder of findings recorded in survivors was within the range of normal background lesions which may be recorded in animals of this strain and age.
Other effects:
no effects observed
Description (incidence and severity):
Testes - detailed examination including sperm staging:

PLEASE REFER TO THE ATTACHED TABLE: "TESTES - DETAILED EXAMINATION INCLUDING SPERM STAGING"
Details on results:
The oral administration of Butyl Hydrogen Maleate to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation period for females) at dose levels of 175, 100 and 30 mg/kg bw/day resulted in treatment-related changes at all dose levels.

Increased salivation was detected soon after dosing for animals of either sex treated with 175 mg/kg bw/day, and this finding was also evident at 100 mg/kg bw/day. Increased salivation is commonly observed following the oral administration of an irritant test item formulation. This was supported by microscopic gastric changes detected during the histopathological examinations. Forestomach hyperkeratosis and squamous cell hypertrophy were evident at 100 and 175 mg/kg bw/day. Focal squamous cell hyperplasia of forestomach was also recorded for animals of either sex treated with 175 mg/kg bw/day. All of these findings were considered to be attributable to the irritant property of the test item. Hyperkeratosis and squamous cell hypertrophy were considered to be a simple reactive change (being within a physiological reaction) to the irritant stimuli of the test item, and hence, deemed not to be adverse. Squamous cell hyperplasia was also evident and was likely to be the reparative change following the tissue injury in the forestomach for animals treated with 175 mg/kg bw/day during the treatment period; therefore, this hyperplastic lesion was deemed to be adverse.

Males treated with 175 mg/kg bw/day showed an increase in bile acids when compared to controls. Increases in liver weights, both absolute and relative to terminal body weight were recorded for animals of either sex treated at all dose levels. Furthermore, histopathological examinations revealed diffuse hepatocellular hypertrophy for animals of either sex treated at all dose levels. Hepatocellular hypertrophy is considered to be a metabolic change and adaptive in nature. Therefore, this finding is considered not to be adverse. For animals showing hepatocellular hypertrophy, single cell death of the hepatocyte was recorded in two males treated with 175 mg/kg bw/day. Single cell death was considered to be caused by the functional overload to the hepatocytes, and therefore, it was deemed to be adverse.

An increase in thyroid weights was noted for males treated with 175 mg/kg bw/day, and histopathological examinations revealed increased incidence and/or severity of thyroid follicular cell hypertrophy for males treated with 175 mg/kg bw/day and females treated at all dose levels. This was considered to be associated with the increased hepatic metabolism of thyroid hormones (T3/T4) due to the hepatocellular hypertrophy.

Therefore the finding was deemed to represent a secondary effect and not to be adverse.

Treatment-related microscopic changes were also evident in the kidneys. Animals of either sex treated with 175 and 100 mg/kg bw/day, and females treated with 30 mg/kg bw/day showed increases in absolute and body weight-relative kidney weights. Microscopic changes were also observed. These consisted of an increased incidence and severity of renal tubular degeneration and regeneration, tubular dilatation, casts, and increased incidence or severity of interstitial mononuclear cell infiltration recorded for animals of either sex treated with 175 mg/kg bw/day. In addition, pelvic urothelial cell hypertrophy was recorded in one female treated with 175 mg/kg bw/day, which was deemed to be a secondary change following the lesions described above. These suggest that there was excessive cellular stress followed by injuries, and hence, were deemed to be adverse. Renal proximal tubular hypertrophy was recorded for animals of either sex treated with 100 and 175 mg/kg bw/day, in a dose-related manner. Since the proximal tubule is the segment having particularly a high metabolic demand in the kidney, such change was considered to be of metabolic nature and of adaptive character, and hence, deemed not to be adverse.

From the findings of this study, a ‘No Observed Effect Level’ (NOEL) could not be established. A ‘No Observed Adverse Effect Level’ (NOAEL) was established at 100 mg/kg bw/day.

No treatment-related effects on reproductive performance or offspring development were detected. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore established at 175 mg/kg bw/day.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The effects detected at 100 and 30 were considered not to represent an adverse effect, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
175 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Incidence and Mean Severity Grade of Main Findings in Stomach (terminal kill)

 

Finding Incidence /

Mean Severity Grade

 

Group 1

 

Group 2

 

Group 3

 

Group 4

 

5 M

5 F

5 M

5 F

5 M

5 F

5 M

5 F

Forestomach, Hyperkeratosis

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

1/1.0

 

1/1.0

 

4/1.0

 

2/1.5

Forestomach, Squamous cell hypertrophy (Increase of mucosal thickness) Incidence/Mean Severity

 

 

0

 

 

0

 

 

0

 

 

0

 

 

1/1.0

 

 

1/1.0

 

 

3/1.0

 

 

2/1.5

Forestomach, Squamous cell hyperplasia, focal Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

0

 

0

 

1/1.0

 

2/1.5

 

Incidence and Mean Severity Grade of Main Findings in Liver (terminal kill)

 

Finding Incidence /

Mean Severity Grade

 

Group 1

 

Group 2

 

Group 3

 

Group 4

 

5 M

5 F

5 M

5 F

5 M

5 F

5 M

5 F

Hepatocellular hypertrophy, diffuse

Incidence/Mean Severity

 

0

 

0

 

2/1.0

 

1/1.0

 

4/1.3

 

5/1.4

 

5/2.0

 

5/1.6

Single cell death, hepatocytes

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

0

 

0

 

2/1.0

 

0

 

Incidence and Mean Severity Grade of Main Findings in Thyroid Gland (terminal kill)

 

Finding Incidence /

Mean Severity Grade

 

Group 1

 

Group 2

 

Group 3

 

Group 4

 

5 M

5 F

5 M

5 F

4 M

5 F

5 M

5 F

Follicular cell hypertrophy

Incidence/Mean Severity

 

1/1.0

 

0

 

0

 

2/1.0

 

1/1.0

 

2/1.0

 

3/1.0

 

3/1.3

 

Incidence and Mean Severity Grade of Main Findings in Kidneys (terminal kill)

 

Finding Incidence /

Mean Severity Grade

 

Group 1

 

Group 2

 

Group 3

 

Group 4

 

5 M

5 F

5 M

5 F

5 M

5 F

5 M

5 F

Tubular hypertrophy, proximal tubules

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

2/1.0

 

3/1.0

 

5/2.0

 

5/1.8

Tubular degeneration/ regeneration Incidence/Mean Severity

 

1/1.0

 

1/1.0

 

0

 

1/1.0

 

2/1.0

 

1/1.0

 

4/1.5

 

5/1.8

Tubular dilatation

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

0

 

0

 

1/1.0

 

4/1.0

Cast(s), hyaline and/or granular

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

0

 

0

 

1/1.0

 

1/2.0

Mononuclear cell focus/foci, interstitium

Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

1/1.0

 

0

 

1/2.0

 

4/1.0

Pelvic urothelial cell hypertrophy Incidence/Mean Severity

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

1/1.0

 

Applicant's summary and conclusion

Conclusions:
The oral administration of Butyl Hydrogen Maleate to rats by gavage, at dose levels of 175, 100 and 30 mg/kg bw/day, resulted in treatment-related changes at all dose levels. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity could therefore not be established.
The effects detected at 100 and 30 were considered not to represent an adverse effect, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day.
There were no treatment-related effects detected on the reproductive parameters investigated, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 175 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 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 the 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 up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 175 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural 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 (during the final week of treatment), and for five selected parental females from each dose group on Day 4 post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Surviving males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post 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 unscheduled deaths related to test item toxicity.

Clinical Observations. Increased salivation and noisy respiration were recorded for animals of either sex treated with 175 mg/kg bw/day, and also some instances for males treated with 100 mg/kg bw/day.

Behavioural Assessment. No treatment-related effects were detected.

Functional Performance Tests. No treatment-related effects were detected.

Sensory Reactivity Assessments. No treatment-related effects were detected.

Body Weight. No adverse effects on body weight change were detected.

Food Consumption. No adverse effects on dietary intake were detected.

Water Consumption. Daily visual inspection of water bottles did not reveal any significant intergroup differences.

Reproductive Performance:

Mating No treatment-related effects were detected in mating performance.

Fertility. No treatment-related effects on fertility were detected.

Gestation Lengths. No treatment-related effects were detected in the length of gestation between control and treated groups.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. No differences in litter size, viability or sex ratio were evident for offspring from treated litters when compared to those from the controls. No toxicologically significant differences in corpora lutea, implantation sites, or pre- and post-implantation losses were detected for females from treated groups when compared to controls.

Offspring Growth and Development. No toxicologically important differences in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters. No treatment-related clinical signs were observed. Post-mortem examinations did not reveal any treatment-related macroscopic findings for offspring.

Laboratory Investigations:

Haematology. No toxicologically significant haematological changes were detected.

Blood Chemistry. An increase in bile acids was noted for males treated with 175 mg/kg bw/day when compared to controls. Pathology:

Necropsy. No treatment-related macroscopic abnormalities were detected.

Organ Weights. Animals of either sex treated at all dose levels showed an increase in liver weights, both absolute and relative to terminal bodyweights.

Animals of either sex treated with 175 and 100 mg/kg bw/day, and females treated with 30 mg/kg bw/day showed an increase in kidney weights, both absolute and relative to terminal body weights, when compared to controls.

Males treated with 175 mg/kg bw/day showed an increase in thyroid weights when compared to controls.

Histopathology. Preliminary histopathology examinations revealed the following treatment-related effects:

STOMACH: Forestomach mucosal squamous cell hypertrophy and hyperkeratosis was evident for animals of either sex treated with 175 and 100 mg/kg bw/day. Focal squamous cell hyperplasia of forestomach was recorded for animals of either sex treated with 175 mg/kg bw/day.

LIVER: A higher incidence and severity of diffuse hepatocellular hypertrophy was recorded for animals of either sex treated at all dose levels when compared to controls. Single cell death of hepatocytes was also observed for two males treated with 175 mg/kg bw/day.

KIDNEYS: Increased incidence/severity of proximal tubular degeneration/regeneration, proximal tubular hypertrophy, tubular dilatation, hyaline and/or granular cast(s), interstitial mononuclear cell focus/foci were recorded at 175 mg/kg bw/day and interstitial mononuclear cell focus/foci was seen at 175 and 100 mg/kg bw/day. Pelvic urothelial hypertrophy was also observed for one female treated with 175 mg/kg bw/day.

THYROID GLANDS: Follicular cell hypertrophy was observed for males treated with 175 mg/kg bw/day and for females from all treatment groups.

Conclusion. The oral administration of Butyl Hydrogen Maleate to rats by gavage, at dose levels of 175, 100 and 30 mg/kg bw/day, resulted in treatment-related changes at all dose levels. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity could therefore not be established.

The effects detected at 100 and 30 were considered not to represent an adverse effect, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day.

There were no treatment-related effects detected on the reproductive parameters investigated, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 175 mg/kg bw/day.