Butyl hydrogen maleate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

other:

Reproductive effects observed:

not specified

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 May 2012 and 06 July 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

no guideline required

Guideline:

other: Please see "any other information on materials and methods" below for details.

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: ~16 weeks
- Weight at study initiation:
males: 379-448g
females: 218-246g
- Fasting period before study: No
- Housing: solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding the animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK, Barrier Maintained Rodent Facility
- Diet (e.g. ad libitum): ad libitum - A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used.
- Water (e.g. ad libitum): ad libitum - Mains drinking water was supplied from polycarbonate bottles attached to the cage
- Acclimation period: The animals were acclimatised for five weeks 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

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered daily, for up to fourteen consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.

Treatment Group Dose Level (mg/kg bw/d) Treatment Volume (ml/kg) Concentration (mg/ml) Males Females
Control 0 4 0 3 3
Low 30 4 7.5 3 3
Intermediate 100 4 25 3 3
High 225* 4 56.3 3 3

*Following the excessive body weight loss in one high dose level male treated with 300 mg/kg bw/day and the early termination of that male on Day 5 of the study (prior to dosing) this dose level was reduced to 225 mg/kg bw/day. Treatment was discontinued for the high dose animals on Day 5 of the study (to act as a ‘wash out’ period) and then recommenced on Day 6 at the adjusted dose level. For the majority of the duration of the treatment period the high dose level of 225 mg/kg bw/day was used therefore it is reported as such.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 d

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
225 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the absence of any toxicity data, preliminary toxicity work was undertaken, treating one male and one female rat at 500 mg/kg bw/day and another one male and one female rat at 100 mg/kg bw/day for three days. Treatment related effects consisted of noisy respiration and body weight loss in animals treated with 500 mg/kg bw/day.
Following preliminary work, animals were allocated to treatment groups as follows:

Treatment Group Dose Level (mg/kg bw/d) Treatment Volume (ml/kg) Concentration (mg/ml) Males Females
Control 0 4 0 3 (1-3) 3 (4-6)
Low 30 4 7.5 3 (7-9) 3 (10-12)
Intermediate 100 4 25 3 (13-15) 3 (16-18)
High 225* 4 56.3 3 (19-21) 3 (22-24)

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made five hours following dosing whenever possible (not at weekends or on public holidays). All observations were recorded.

Body Weight
Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

Food Consumption
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was measured and recorded daily for each cage group

Sacrifice and pathology:

On completion of the dosing period, all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination. Animals that died during the study were also necropsied. Any tissues showing macroscopic abnormalities were preserved in buffered 10% formalin. These will be discarded following issue of the final report.

Statistics:

Data were processed to give individual animal/group mean values, standard deviations and incidence of findings where appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Mortality:

mortality observed, treatment-related

Description (incidence):

Please see "details on results" below for further information.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

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

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Please see "details on results" below for further information.

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality
One male (No. 19) treated with 300 mg/kg bw/day was killed in extremis on Day 5 of the study (prior to dosing) due to excessive body weight loss.
There were no other unscheduled deaths during the study.

Clinical Observations
The male treated with 300 mg/kg bw/day (No. 19) that was killed in extremis had noisy respiration between Days 1 and 5, ataxia on Day 4 and hunched posture, lethargy, decreased respiratory rate on Day 5 of the study.
Following the reduction of the high dose level animals of either sex treated with 225 mg/kg bw/day showed incidents of noisy respiration, increased salivation and one female from this treatment group also showed laboured respiration and a decreased respiratory rate.
No such effects were evident in animals of either sex treated with 100 or 30 mg/kg bw/day.

Body Weight
One male (No. 19) treated with 300 mg/kg bw/day showed an excessive body weight loss on Day 4. The remaining high dose animals of either sex also showed body weight losses or remained static. Following the reduction of the high dose level, animals of either sex treated with 225 mg/kg bw/day continued to show incidents of reduced body weight gains and a subsequent reduction in overall body weight gains.
Overall body weight gain was also reduced in males treated with 100 mg/kg bw/day. No toxicological significant effects were evident in females treated with 100 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Food Consumption
Males treated with 225 mg/kg bw/day showed a reduction in food consumption and food efficiency between Days 1 and 11 of the study. No adverse effect on food consumption was evident in the high dose females, however females from this treatment group showed a reduction in overall food efficiency.
No such effects were evident in animals of either sex treated with 100 or 30 mg/kg bw/day.

Water Consumption
Daily measurements of water consumption revealed an increase in water intake in animals of either sex treated with 225 and 100 mg/kg bw/day.
No toxicologically significant effects were detected in animals of either sex treated with 30 mg/kg bw/day.

Necropsy
The male treated with 300 mg/kg bw/day that was killed in extremis had sloughing on the non-glandular region of the stomach and gaseous distension of the small and large intestines at necropsy.
One female treated with 225 mg/kg bw/day had gaseous distension of the small and large intestines and another female from this treatment group had reddened lungs with dark foci at necropsy.
No macroscopic findings were recorded in males treated with 225 mg/kg bw/day or animals of either sex treated with 100 or 30 mg/kg bw/day.

Dose descriptor:

other: Suitable high dose level for use on future toxicity studies.

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

DISCUSSION

The oral administration of Butyl Hydrogen Maleate to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day (reduced to 225 mg/kg bw/day on Day 5) resulted in treatment related effects in the high dose animals and animals of either sex treated with 100 mg/kg bw/day.

One male from the high dose group was killed in extremis on day 5 due to excessive body weight loss and clinical observations consisting of noisy respiration, ataxia, hunched posture, laboured respiration and a decreased respiratory rate.

Following the reduction of the high dose level treatment at 225 mg/kg bw/day resulted in adverse effects seen in the surviving animals consisting of reductions in body weight development and dietary intake together with an increase in water consumption. Clinical observations were confined to increased salivation, noisy and laboured respiration and a decreased respiratory rate. Macroscopic examinations revealed gaseous distension of the small and large intestines in one female and reddened lungs with dark foci in another female.

Observations at 100 mg/kg bw/day were less severe than the high dose level and consisted of a reduction in body weight gains in male rats only and an increase in water consumption in animals of either sex.

These findings indicate that a dose level of 225 mg/kg bw/day would prove unsuitable for use on longer term oral administration studies. The findings recorded at 100 mg/kg bw/day were considered not to represent an adverse effect of treatment and on this basis the following dose levels are recommended for use in a repeat dose/reproduction screening toxicity study: 30, 100 and 175 mg/kg bw/day.

Conclusions:

The oral administration of Butyl Hydrogen Maleate to rats for a period of up to fourteen consecutive days at dose levels of 300 (reduced to 225 mg/kg bw/day on Day 5 of the study), 100 or 30 mg/kg bw/day resulted in treatment related effects at the high dose level and at 100 mg/kg bw/day. The suitable high dose level for use on future toxicity studies is considered to be 175 mg/kg bw/day.

Executive summary:

The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for up to fourteen consecutive days, at dose levels of 30, 100 and 225 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

The oral administration of Butyl Hydrogen Maleate to rats for a period of up to fourteen consecutive days at dose levels of 300 (reduced to 225 mg/kg bw/day on Day 5 of the study), 100 or 30 mg/kg bw/day resulted in treatment related effects at the high dose level and at 100 mg/kg bw/day. The suitable high dose level for use on future toxicity studies is considered to be 175 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 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

Test material information

Constituent 1

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

Specific details on test material used for the study:

Sponsor's identification : Butyl Hydrogen Maleate Description : pale yellow liquid
Purity : >95%
Batch 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 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
- 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

Vehicle:

arachis oil

Details on 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.

Details on mating procedure:

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.

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

Duration of test:

57d

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control treatment group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low treatment group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Intermediate treatment group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High treatment group

No. of animals per sex per dose:

12

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.

Examinations

Maternal examinations:

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

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
Pathology
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.

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 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:
Seminal vesicles, Epididymides, Ovaries, Uterus (weighed with Cervix) and Pituitary (post fixation)

Histopathology
Samples of the following tissues from 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)
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
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].

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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).

Fetal examinations:

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.

Statistics:

Please refer to the section below "Any other information on materials and methods"

Indices:

Please refer to the section below "Any other information on materials and methods"

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

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:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effects on body weight change were detected for females during the pre-pairing, gestation or lactation phases of the study.

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 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 ATTACHED TABLE: "GROUP MEAN FOOD CONSUMPTION"

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant differences were detected in the haematological parameters investigated.

PLEASE REFER TO THE ATTACHED ATBLE: "GROUP MEAN HAEMATOLOGICAL VALUES"

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 ATBLE: "GROUP MEAN BLOOD CHEMICAL VALUES"

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioural Assessments
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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-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).

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):

There were no treatment-related macroscopic abnormalities detected.

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed the following treatment-related changes:

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)"

Histopathological findings: neoplastic:

effects observed, non-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.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Not applicable, as rats do not abort

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No toxicologically significant differences in corpora lutea, implantation sites, pre- and post-implantation losses or sex ratio were detected for females from treated groups when compared to controls.

PLEASE REFER TO THE ATTACHED TABLE: "GROUP MEAN IMPLANTATION LOSSES AND SURVIVAL INDICES VALUES"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No toxicologically important differences in litter size or viability were evident for offspring from treated litters when compared to those from the controls.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in the length of gestation between control and treated groups. Gestation lengths of 22 to 23 ½ days were recorded for all pregnant females.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on fertility were detected for treated animals when compared to controls.

There were two non-pregnant females treated with 100 mg/kg bw/day and one non- pregnant female from the 175 mg/kg bw/day dose group. Two control females also did not achieve pregnancy following mating. Non-pregnancies are low incidence findings in reproductive studies of this type and as there were two non-pregnancies observed in the control group, the absence of pregnancy in the animals at 100 and 175 mg/kg bw/day was considered to be unrelated to treatment.

PLEASE REFER TO THE ATATCHED TABLE: "SUMMARY INCIDENCE OF MATING PERFORMANCE, FERTILITY AND GESTATION LENGTH"

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were detected in mating performance. The majority of animals mated within the first five days of pairing.

A statistically significant reduction in implantation sites, number of offspring born, and litter size on Day 1 and Day 4 of lactation was observed for females treated with 100 mg/kg bw/day when compared to controls. In the absence of a dose-related response, these reductions were considered to be of no toxicological importance.

A significantly lower number of implantation sites were also noted for females treated with 30 mg/kg bw/day when compared to controls. The significance achieved was minimal (p<0.05) and in isolation, this finding was considered to have arisen incidentally.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Necropsy-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.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

175 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: 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.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No toxicologically important differences in litter weights, offspring weights or surface righting were detected for litters from treated groups when compared to control litters.

PLEASE REFER TO THE ATATCHED ATBLE: "GROUP MEAN LITTER SIZE AND LITTER WEIGHTS"

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

In total ten females from control, all twelve females treated with 30 mg/kg bw/day, ten females treated with 100 mg/kg bw/day and eleven females treated with 175 mg/kg bw/day gave birth to a live litter and successfully reared young to Day 5 of age

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

Post-mortem examinations did not reveal any treatment-related macroscopic findings for offspring at terminal kill.

PLEASE REFER TO THE ATATCHED TABLE: "SUMMARY INCIDENCE OF NECROPSY FINDINGS"

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Necropsy-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

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

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

 

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:

MatingNo 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-mortemexaminations 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.