4,4'-isopropylidenebis[2-allylphenol]

Endpoint summary

Description of key information

In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) the NOAEL for systemic toxicity was found to be 85 mg/kg/day. Effects were noted at 250 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 19 June 2014 and 19 May 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

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:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 295 to 376g, the females weighed 200 to 242g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. There were no deviations from the target range for temperature but values for relative humidity exceeded the target range on a number of occasions; these deviations were considered not to have affected the purpose or integrity of the study (see deviation from Study Plan).

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Justification
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.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least fourteen days when stored at room temperature in the dark. Formulations were initially prepared weekly and then on a fortnightly basis. Formulations were stored at approximately 4 °C in the dark prior to use.

Representative samples of the test item formulation were taken on four occasions during the study and analyzed for concentration of 4,4’-isopropylidenebis[2-allylphenol] (TK 11907) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 101-109% of the nominal concentration confirming the accuracy and suitability of the formulation procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external calibration. An aliquot, 100 mg of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile; this was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, samples solutions were further diluted with acetonitrile to achieve the working concentration.

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

Preparation of Linearity samples
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.067 mg/mL by serial dilution covering the concentration range 0 to 0.1601 mg/mL.

Instrumental setup
HPLC: Agilent Technologies 1200, incorporating autosampler and work station
Column: Prodigy 3 µ ODS (150 x 4.6 mm id)
Mobile phase: Acetonitrile: water (80:20 v/v)
Flow rate: 1 mL/min
UV detector wavelength: 283 nm
Injection volume: 10 µL
Retention time: ~ 3 mins

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

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

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration cure to the data was 1.000 and considered to be acceptable.

Accuracy
The fortified samples of arachis oil BP were found to have a recovery value of ± 10 % of the fortification.

Test item formulation
The formulations investigated during the study were found to comprise test item in the range ot 101 % to 109 % and thus the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 14 days at room temperature due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and arachis oil BP as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

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

Duration of treatment / exposure:

Up to eight weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
85, 250 and 750/500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Twelve per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were chosen, in collaboration with the Sponsor, based on available toxicity data including a rat fourteen day range finder toxicity study (Harlan Study Number: 41401332). In this preliminary study, a dosage of 1000 mg/kg bw/day was associated with one death, clear initial effects on body weight and food and sloughing of the glandular region of the stomach. Findings at 500 mg/kg bw/day were unremarkable except for one male with sloughing of the glandular region of the stomach. It was concluded that the high dosage on this main OECD 422 study should therefore lie between 500 and 1000 mg/kg bw/day.

Due to adverse body weight effects for some males at 750 mg/kg bw/day during Week 1 of the study, the high dosage was reduced to 500 mg/kg bw/day from Week 2 of the study.

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

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

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. 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:

None

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.


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


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

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

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

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

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


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

The following parameters were observed:

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

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. Additional bodyweights were recorded for control animals during Days 14 and 15 and for high dosage animals during the second week of treatment for the monitoring of animal health. These unscheduled weights are retained in the raw data but are not presented in this report. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

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

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


Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.

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


Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 08:30 and as late as possible at weekends and public holidays. The following was recorded for each female:

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


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

For each litter the following was recorded:

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


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

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

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

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

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

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

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

Sacrifice and pathology:

Pathology
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to 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). The corpora lutea were also counted.

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


Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group.
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

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


Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated.
Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides•, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes* , Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes•, Liver, Thymus, Lungs (with bronchi) #, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina.

Tissues shown below were preserved from all remaining animals:
Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Testes, Uterus/Cervix, Vagina, Mammary gland.

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

Tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 750 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown from the remaining control and 750 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 750 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Following the results of the initial histopathological examinations of control and high dosage animals, microscopic evaluation of the tissues was extended to include the liver and kidneys to the low and intermediate dosage groups.

Microscopic examination was conducted by the Study Pathologist (Jeffrey Wilson at Propath GmbH, Muttenzerstrasse 30, 4133 Pratteln, Switzerland).

Other examinations:

None specified

Statistics:

See below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See results

Mortality:

mortality observed, treatment-related

Description (incidence):

See results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See results

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See results

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

effects observed, treatment-related

Description (incidence and severity):

See results

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: neoplastic:

no effects observed

Details on results:

Adult Reponses
Mortality
There were two unscheduled deaths on the study.

At 750 mg/kg bw/day, male number 76 showed excessive weight loss during Week 1. Despite the reduction of dosage to 500 mg/kg bw/day for Week 2, this animal subsequently showed hunched posture and emaciation prior to dosing on Day 9 and was killed for animal welfare considerations. Clinical signs prior to this had been restricted to increased post-dosing salivation. Necropsy revealed dark liver, enlarged kidneys with a dark area, enlarged stomach with sloughing of the non-glandular region, enlarged adrenals and small seminal vesicles.

At 750/500 mg/kg bw/day, male number 84 was found dead prior to dosing on Day 10 of the study. Clinical signs prior to this had been restricted to increased post-dosing salivation but the animals had also shown notable weight loss during Week 1 of the study. Necropsy revealed an enlarged stomach with sloughing and raised limiting ridge, enlarged adrenals and mesenteric lymph nodes and small seminal vesicles.

Although both of these decedents died while receiving a dosage of 500 mg/kg bw/day, it is considered that these deaths were most likely to be attributable to the previous treatment at 750 mg/kg bw/day.


Clinical Observations
With the exception of one male at 85 mg/kg bw/day, all surviving treated animals showed increased post-dosing salivation at some stage of the study, the incidence of this finding tending to increase with dosing. Increased post-dosing salivation is frequently observed when a slightly unpalatable or irritant test item is administered via the oral gavage route and is considered to reflect distaste of the dosing formulations rather than any adverse systemic effect of treatment.

In addition to this increased salivation, one male at 250 mg/kg bw/day and two males and three females at 750/500 mg/kg bw/day showed noisy respiration. This finding most probably represents accidental inspiration of the test item formulations during the dosing procedure and at the incidence and duration observed on this study was considered not to represent a systemic effect of treatment.

At 750/500 mg/kg bw/day one male (number 75) showed staining of the ano-genital region during Days 18 to 22.

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


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

For females at 750/500 mg/kg bw/day, the group mean value for the last 20% mobile activity was higher than control and attained statistical significance; however, the data were not particularly suited to statistical analysis and this marginal difference from control was considered to be of no toxicological significance.


Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores.

Body Weight
Males

At 750 mg/kg bw/day, males showed a mean body weight loss during the first week of treatment resulting in a statistically significantly lower mean body weight compared to control on Day 8. There was wide variation in the degree of individual body weight losses observed, with body weight losses being particularly marked for a few animals; however, while body weight loss was evident for seven of the twelve males, body weight gain for the remaining five males was similar to control. Following the reduction of the dosage to 500 mg/kg bw/day, mean body weight gains were generally lower than control but these weekly differences were not marked and statistical significance was only attained during Week 4 (Days 22 to 29). During this week, lower body weight gains for all treated males attained statistical significance when compared with control but showed no dosage-relationship; these differences may reflect particularly good performance by the control animals rather than a treatment related effect. The slightly lower body weight gains apparent after the reduction of the dosage to 500 mg/kg bw/day meant that mean body weights continued to be statistically significantly lower than control to termination and lower overall mean body weight gain (Days 1-43) was also statistically significant. The differences in overall mean body weight gain from the reduction of the dosage to termination (Days 8-43) were only slightly lower than control and differences failed to attain statistical significance.

At 85 and 250 mg/kg bw/day, body weight gains were generally similar to control and did not appear to be adversely affected by treatment. As previously discussed, difference in body weight gain from control during Week 4 at these dosages did attain statistical significance but were considered to be unrelated to treatment.


Females
There was no clear adverse effect of treatment on body weight gain of females during the pre-pairing, gestation and lactation phases of the study at 85, 250 and 750/500 mg/kg bw/day.

For females at 750 mg/kg bw/day, a slight mean body weight loss was apparent during the first week of treatment; however mean body weight gain of control were slight during this period. The females on this study design are no longer on a steep growth curve and small body weight fluctuations, including slight body weight losses, are frequently observed. While body weight losses were apparent for males at this dosage during Week 1, the degree of weight losses for females was unremarkable and no association with treatment was considered to be proven.

Food Consumption
At 750 mg/kg bw/day, food consumption for males was lower than control during the first week of treatment and continued to be lower for the second week despite the lowering of the dosage to 500 mg/kg bw/day. Food consumption for these males during the post-pairing phase of the study was similar to control.

There was no obvious effect on food consumption for males at 85 and 250 mg/kg bw/day.

Other than a suggestion of lower food consumption during the first week of treatment at 750 mg/kg bw/day, food intake for females at 85, 250 and 500 mg/kg bw/day was unaffected by treatment during the pre-pairing, gestation and lactation phases of the study.


Food Conversion Efficiency
The value of assessing food conversion efficiency where a group mean body weight loss has occurred is equivocal.

For males at 750 mg/kg bw/day food conversion efficiency during the first week of treatment was inferior to control reflecting the mean body weight loss apparent for these animals. Following the lowering of the dosage to 500 mg/kg bw/day, food conversion efficiency did not appear to be adversely effected by treatment. For females at 750 mg/kg bw/day, food conversion efficiency was also negative during the first week of treatment, although the difference from control was slight and no clear association with treatment was considered proven.

At 85 and 250 mg/kg bw/day, food conversion efficiency for both sexes during the pre pairing phase and males during the post-mating phase of the study was considered to be unaffected by treatment.

Water Consumption
At 750/500 mg/kg bw/day, mean water consumption during the pre-pairing period was higher than control from Day 3 for males and Day 2 for females. For females, water intake was also higher during Day 1 but intergroup differences indicated that this difference probably reflected normal biological variation rather than an effect of treatment.

At 250 mg/kg bw/day, mean water consumption during the pre-pairing period was higher than control from Day 2 for both sexes.

At 85 mg/kg bw/day, differences in water consumption for either sex during the pre-pairing phase did not show any consistency that indicated an effect of treatment.


Reproductive Performance
Mating and Fertility
There were no treatment-related effects on mating performance as assessed by pre-coital interval; however at 750/500 mg/kg bw/day the number of matings that resulted in a pregnancy was clearly lower than the control and the other treated groups.


Gestation Length
Gestation lengths were between 22 and 23½ days and were considered to have been unaffected by maternal treatment.


Litter Responses
One control and five females receiving 750/500 mg/kg bw/day were found to be non-pregnant. All pregnant females successfully gave birth and reared their offspring to Day 5 post partum; in total there were 11, 12, 12 and 7 litters available for assessment in the control, 85, 250 and 750/500 mg/kg bw/day groups respectively.

Offspring Litter Size, Sex Ratio and Viability
There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 85, 250 and 750/500 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were essentially similar or slightly superior to controls and appeared unaffected by maternal treatment at 85, 250 and 750/500 mg/kg bw/day. Offspring performance during assessment of surface righting appeared to be unaffected by maternal treatment.

The clinical sign and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence or distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 85, 250 and 750/500 mg/kg bw/day.

Laboratory Investigations
Hematology
For males at 750/500 mg/kg bw/day, lower hemoglobin, erythrocyte count and hematocrit attained statistical significance when compared to control; individual values for these parameters were outside the historical control range for two of the five males assessed at this dosage. Additionally at 750/500 mg/kg bw/day, activated partial thromboplastin time was also statistically significantly lower than control but individual values were outside the historical control range for only one of the treated males.

For males at 250 mg/kg bw/day, lower erythrocyte count also attained statistically significance when compared with control but all individual values were within the historical control range.

For males at 85 mg/kg bw/day, higher mean cell volume and mean cell hemoglobin attained statistical significance when compared with control, but all individual values for these treated animals were within the historical control range and, in the absence of any similar increase at higher dosages, this finding was considered to be incidental and unrelated to treatment.

For females at all dosages, there were no statistically significant differences from control for any of the hematology parameters measured.


Blood Chemistry
At 250 and 750/500 mg/kg bw/day for both sexes, higher alanine aminotransferase levels attained statistical significance when compared with control. For females, mean values showed a dosage relationship and, at both dosages, the majority of individual values exceeded the historical control range. For males, the mean values did not show a dosage relationship and, while the majority of individual values at 250 mg/kg bw/day exceeded the historical control range only two individual values exceeded this historical range at 750/500 mg/kg bw/day. At 85 mg/kg bw/day, higher alanine aminotransferase for females also attained statistical significance when compared with control but all individual values were within the historical control range.

At 250 and 750/500 mg/kg bw/day higher aspartate aminotransferase levels for females attained statistical significance when compared with control; the majority of individual values at 750/ 500 mg/kg bw/day exceeded the historical control range but only one individual value exceeded this historical range at 250mg/kg bw/day.

At 250 and 750/500 mg/kg bw/day, total bilirubin levels for both sexes were higher than control with differences attaining statistical significance. For males, mean values showed a dosage relationship and, at both dosages, the majority of individual values exceeded the historical control range. For females, the mean values did not show a dosage relationship and, while all individual values at 250 mg/kg bw/day exceeded the historical control range only two individual values exceeded this historical range at 750/500 mg/kg bw/day.

At 250 and 750/500 mg/kg bw/day, higher total cholesterol levels for males attained statistical significance when compared with control but only two individual values at each dosage exceeded the historical control range. Higher total cholesterol levels also attained statistical significance for males at 85 mg/kg bw/day but all individual values were within the historical control range.

At 750/500 mg/kg bw/day, blood urea levels for both sexes were statistically significantly higher than control; the majority of individual values for females exceeded the historical control range but only one individual value for males exceeded this historical range.

Additionally for males at 750/500 mg/kg bw/day, lower total protein and albumin levels attained statistical significance when compared with control; however there was no accompanying statistically significant difference for mean albumin/globulin ratio and, for both parameters, the majority of individual values for these treated animals were within the historical control range.

Pathology - Necropsy
Offspring
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicated any effect of maternal treatment at 85, 250 and 750/500 mg/kg bw/day.


Adults
At 750/500 mg/kg bw/day, the majority of surviving males showed some macroscopic kidney findings, such as enlargement, pallor, or mottled appearance at terminal necropsy. At 250 mg/kg bw/day, one male showed enlargement of the kidneys and another pale kidneys at terminal necropsy.

There were no macroscopic abnormalities considered to be associated with treatment for both sexes at 85 mg/kg bw/day or for females at 250 and 750/500 mg/kg bw/day. One female at 750/500 mg/kg bw/day showed fluid filled increased pelvic space of the left kidney but this finding is occasionally observed in rats of this strain and, in isolation, was considered incidental and unrelated to treatment.

One male at both 250 and another male at 750/500 mg/kg bw/day were observed with unilateral small seminal vesicles at terminal necropsy; both animals induced pregnancy in their respective female partners and in isolation this finding was considered incidental and unrelated to treatment.


Organ Weights
At 250 and 750/500 mg/kg bw/day, there was an increase in absolute and body weight relative kidney weights for males compared with control, although differences only attained statistical significance at the highest dosage. For this organ, body weight relative values are considered to be the best indicator of toxicological effect, and the majority of individual body weight values at 250 and all body weight values at 750/500 mg/kg bw/day exceeded the historical control range compared to only one body weight value in the control group.

For females at 750/500 mg/kg bw/day, absolute and body weight relative kidney weights were also statistically significantly higher than control and majority of individual body weight values for these treated animals exceeded the historical control range.

At 250 and 750/500 mg/kg bw/day, there was an increase in absolute and body weight relative liver weights for males compared with control, with differences attaining statistical significance. Again for this organ, body weight relative values are considered to be the best indicator of toxicological effect and the majority of individual body weight relative values at 250 mg/kg bw/day and all body weight relative values at 750/500 mg/kg bw/day exceeded the historical control range; one body relative weight value in the control group was below this historical range.

For females at 750/500 mg/kg bw/day, absolute and body weight relative liver weights were also statistically significantly higher than control but only one individual body weight relative value for these treated animals exceeded the historical control range.

For males at 85 mg/kg bw/day, absolute and body weight relative liver weights were also higher than control but differences failed to attain statistical significance. Only two body weight relative values for these treated animals exceeded the historical control range and, in the absence of any histopathological change for the liver at this dosage, this finding was considered to be of no toxicological significance.

At 250 mg/kg bw/day and 750/500 mg/kg bw/day, lower absolute and body weight relative heart weights for males attained statistical significance compared with control, but, with the exception of one individual absolute heart weight at 750/500 mg/kg bw/day, all individual values for these treated animals were within the historical control range.

For males at all dosages, lower absolute and body weight relative pituitary weights attained statistical significance compared with control, but mean values showed no consistent dosage relationship. The majority of individual values were within the historical control range and, in the absence of any supporting histopathological change, this finding was considered incidental and unrelated to treatment.

At 85, 250 and 750/500 mg/kg bw/day, lower absolute and body weight relative thymus weights for females attained statistical significance when compared to control, however all individual values for treated animals were outside the historical control range. At 250 mg/kg bw/day and 750/500 mg/kg bw/day, lower absolute and body weight relative thymus weights for males also attained statistical significance when compared to control; mean values showed no dosage relationship and only one control and one 250 mg/kg bw/day body weight relative value were outside the historical control range.

At 750/500 mg/kg bw/day, lower absolute and body weight relative prostate and seminal vesicles weights attained statistical significance when compared to control but all individual values for these treated animals were with the historical control range.

At 250 mg/kg bw/day and 750/500 mg/kg bw/day, higher absolute and body weight relative spleen weights for males attained statistical significance when compared with control, although only mean body weight relative values showed a dosage relationship. At 250 mg/kg bw/day, two individual absolute values and three body weight relative vales exceeded the historical control range, while at 750/500 mg/kg bw/day three individual absolute values and two body weight relative values exceeded this historical range.

Histopathology
Microscopic findings considered to be treatment related were observed for the liver and kidneys.

For the liver, diffuse midzonal/centrilobular hypertrophy at minimal degree were recorded in 1/5 males at 250 mg/kg bw/day and at minimal or slight in 6/7 males and 1/5 females at 750/
500 mg/kg bw/day.

For the kidneys, cortical hyaline droplets at minimal or slight degree were recorded in 4/5 control males, at minimal to moderate in all 5/5 males at 85 mg/kg bw/day, at slight or moderate in 4/5 males at 250 mg/kg bw/day and at minimal or slight degree in 5/10 males at 750/
500 mg/kg bw/day. Tubular dilation with atrophic epithelium was recorded at minimal to moderate degree in 4/5 males at 250 mg/kg bw/day and at slight to very severe in 10/10 males at 750/500 mg/kg bw/day and at minimal or slight degree in 2/5 females at 250 mg/kg bw/day and at slight degree in 2/5 females at 750/500 mg/kg bw/day. Hyaline/granular casts at minimal to moderate degree were noted in 5/5 males at 250 mg/kg bw/day and at moderate to severe degree in 8/10 males at 750/500 mg/kg bw/day and at slight degree in 1/5 males at 85 mg/kg bw/day, minimal in 2/5 females at 250 mg/kg bw/day and at minimal to moderate in 2/5 females at 750/500 mg/kg bw/day. Granulolymphocytic inflammatory infiltrate at minimal degree were seen in 1/5 control males and 1/5 males at 85 mg/kg bw/day, at minimal or slight in all 5/5 males at 250 mg/kg bw/day and at slight or moderate degree in all 10/10 males at 750/500 mg/kg bw/day and at minimal degree in 3/5 females at 250 mg/kg bw/day and at minimal to slight in 2/5 females at 750/500 mg/kg bw/day. Corticomedullary tubular basophilia was recorded at minimal degree in 2/5 control males and 4/5 males at 85 mg/kg bw/day, at slight to severe in all 5/5 males at 250 mg/kg bw/day and at moderate to severe in 10/10 males at 750/500 mg/kg bw/day and at minimal to moderate in 4/5 females at 250 mg/kg bw/day and 4/5 females at 750/500 mg/kg bw/day. Additionally papillary necrosis at slight or moderate degree was noted in both decedent males at 750/500 mg/kg bw/day.

Testes morphology did not reveal any disturbances of spermatogenic staging.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in Han Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.

Dose descriptor:

NOAEL

Effect level:

85 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity based on nephrotoxicity

Critical effects observed:

not specified

Body Weight

Males  

Group		1	2	3	4
Dosage (mg/kg bw/day)		0 (Control)	85	250	700/500
Days 0-43	Mean SD N	84.6 14.9 12	80.0 21.4 12	74.3 20.1 12	54.3** 21.9 10
Days 8-43	Mean SD N	68.3 13.0 12	62.3 15.8 12	57.6 18.7 12	57.1 23.3 10

** P<0.01

Conclusions:

Nephrotoxicity at 250 mg/kg bw/day and at 750/500 mg/kg bw/day precluded these dosages from representing a No Observed Adverse Effect Level (NOAEL) for systemic toxicity therefore the NOAEL for systemic toxicity was considered to be 85 mg/kg bw/day.

As lower pregnancy rate was observed at 750/500 mg/kg bw/day, the clear No Observed Effect Level (NOEL) for reproduction was considered to be 250 mg/kg bw/day. The lower pregnancy rate at 750 mg/kg bw/day may have been a consequence of adverse adult toxicity apparent during the pre-pairing phase. Despite this adverse toxicity for the parental animals, there was no effect on their litters and the NOEL for the survival, growth and development of the offspring was at least 500 mg/kg bw/day.

Executive summary:

Introduction

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

 

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

 

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 85, 250 and 750/500 mg/kg bw/day. The high dosage of 750 mg/kg bw/day was lowered to 500 mg/kg bw/day from Week 2. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group 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 assessed for five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

Surviving adult males were terminated on Day 43 or Day 44, followed by the termination of all surviving 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 two unscheduled deaths on the study, both at 750/500 mg/kg bw/day.

 

Male number 76 showed excessive weight loss during Week 1 and, despite the reduction of dosage to 500 mg/kg bw/day was killed on Day 9 after showing hunched posture and emaciation. Necropsy revealed dark liver, enlarged kidneys with a dark area, enlarged stomach with sloughing of the non-glandular region, enlarged adrenals and small seminal vesicles.

 

Male number 84 showed notable weight loss during Week 1 and was found dead prior to dosing on Day 10 of the study. Necropsy revealed enlarged stomach with sloughing and raised limiting ridge, enlarged adrenals and mesenteric lymph nodes and small seminal vesicles.

 

Clinical Observations

With the exception of one male at 85 mg/kg bw/day, all surviving treated animals showed increased post-dosing salivation at some stage of the study; additionally one male at 250 mg/kg bw/day and two males and three females at 750/500 mg/kg bw/day showed noisy respiration. 

At 750/500 mg/kg bw/day one male showed staining of the ano-genital region during Days 18 to 22.

 

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters at 85, 250 or 750/500 mg/kg bw/day.

 

Functional Performance Tests

There were no changes in functional performance considered to be related to treatment at 85, 250 or 750/500 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores.

 

Body Weight

At 750 mg/kg bw/day, males showed a mean body weight loss during the first week of treatment resulting in statistically significantly lower mean body weight on Day 8. While the majority of animals showed body weight loss, which was quite marked for a few animals, five of the twelve males showed similar body weight gain as control. Following the lowering of the dosage to 500 mg/kg bw/day, mean body weights continued to be statistically significantly lower than control to termination although overall mean body weight gain during this period (Days 8-43) was only slightly lower than control.

 

There was no effect of treatment on body weight gain for both sexes at 85 and 250 mg/kg bw/day and for females at 750/500 mg/kg bw/day during the study (including pre-pairing, gestation and lactation phases for females).

 

Food Consumption

At 750 mg/kg bw/day, lower food consumption for males was apparent during the first week and, to a lesser extent, the second week of the study. For females at this dosage there was a suggestion of lower food consumption during the first week of treatment.

There was no effect of treatment on food consumption for both sexes at 85 and 250 mg/kg bw/day during the study (including pre-pairing, gestation and lactation phases for females).

 

Food Conversion Efficiency

For males at 750 mg/kg bw/day food conversion efficiency during the first week of treatment was inferior to control reflecting the mean body weight loss apparent for these animals. 

 

There was no clear effect of treatment on food conversion efficiency for both sexes at 85 and 250 mg/kg bw/day and for females at 750/500 mg/kg bw/day during the study (including pre-pairing, gestation and lactation phases for females).

 

 

Water Consumption

At 750/500 mg/kg bw/day, mean water consumption during the pre-pairing period was higher than control from Day 3 for males and Day 2 for females.

 

At 250 mg/kg bw/day, mean water consumption during the pre-pairing period was higher than control from Day 2 for both sexes. 

 

There was no effect of treatment on water consumption for both sexes at 85 mg/kg bw/day during the study (including pre-pairing, gestation and lactation phases for females).

 

 

Reproductive Performance

Mating and Fertility

At 750/500 mg/kg bw/day, the number of matings that resulted in a pregnancy was clearly lower than the control and the other treated groups although mating performance, as assessed by pre-coital interval, was unaffected.

 

There was no effect of treatment on mating and fertility at 85 and 250 mg/kg bw/day during the study.

 

 

Gestation Lengths

Gestation lengths were considered to have been unaffected by maternal treatment at 85, 250 or 750/500 mg/kg bw/day.

 

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Numbers of corpora lutea and implantations, pre- and post implantation losses, litter size at birth/Day 1, subsequent offspring survival to Day 4 and sex ratio were unaffected by maternal treatment at 85, 250 or 750/500 mg/kg bw/day.

 

 

Offspring Growth and Development

Offspring body weight and assessment of surface righting on Day 1 and subsequent body weight gain to Day 4post partumwere unaffected by maternal treatment at 85, 250 or 750/500 mg/kg bw/day.

 

Clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed and the incidence/distribution of these observations did not indicate any effect of maternal treatment on offspring development at 85, 250 or 750/500 mg/kg bw/day.

 

 

Laboratory Investigations

Hematology

At 750/500 mg/kg bw/day, hemoglobin, erythrocyte count and hematocrit for males were statistically significantly lower than control.

 

At 250 mg/kg bw/day, erythrocyte count for males was statistically significantly lower than control.

 

There was no adverse effect of treatment on haematology parameters for both sexes at 85 mg/kg bw/day and for females at 250 or 750/500 mg/kg bw/day.

 

 

Blood Chemistry

At 250 and 750/500 mg/kg bw/day alanine aminotransferase levels were statistically significantly higher than control for both sexes. Additionally for females at these dosages, higher aspartate aminotransferase levels attained statistical significance compared to control.

 

At 250 and 750/500 mg/kg bw/day, total billirubin levels for both sexes were statistically significantly higher than control with differences attaining statistical significance.

 

At 250 and 750/500 mg/kg bw/day, higher total cholesterol levels for males attained statistical significance when compared with control but only two individual values at each dosage exceeded the historical control range. 

 

At 750/500 mg/kg bw/day, blood urea levels for both sexes were statistically significantly higher than control.

 

 

Pathology

Necropsy

At 750/500 mg/kg bw/day, the majority of surviving males showed some macroscopic kidney findings, such as enlargement, pallor, mottled appearance. 

 

At 250 mg/kg bw/day, one male showed enlargement of the kidneys and another pale kidneys at terminal necropsy. 

 

There were no macroscopic abnormalities considered to be associated with treatment for both sexes at 85 mg/kg bw/day or for females at 250 and 750/500 mg/kg bw/day.

 

 

Organ Weights

For males at 250 and both sexes at 750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative kidney weights compared with control.

 

For males at 250 and both sexes at 750/500 mg/kg bw/day, there was a statistically significant increase in absolute and body weight relative liver weights compared with control.

 

For females at 750/500 mg/kg bw/day, absolute and body weight relative liver weights were also statistically significantly higher than control but only one individual body weight values for these treated animals exceeded the historical control range.    

 

At 85, 250 and 750/500 mg/kg bw/day, lower absolute and body weight relative thymus weights for females attained statistical significance when compared to control with values showing a consistent dosage relationship; however all individual values for treated animals were outside the historical control range. At 250 mg/kg bw/day and 750/500 mg/kg bw/day, lower absolute and body weight relative thymus weights for males also attained statistical significance when compared to control; mean values showed no dosage relationship and only one control and one 250 mg/kg bw/day body weight relative value were outside the historical control range.

 

At 750/500 mg/kg bw/day, lower absolute and body weight relative prostate and seminal vesicles weights attained statistical significance when compared to control but all individual values for these treated animals were with the historical control range.

 

At 250 mg/kg bw/day and 750/500 mg/kg bw/day, higher absolute and body weight relative spleen weights for males attained statistical significance when compared with control, although only mean body weight relative values showed a dosage relationship. At 250 mg/kg bw/day, two individual absolute values and three body weight relative vales exceeded the historical control range, while at 750/500 mg/kg bw/day three individual absolute values and two body weight relative values exceeded this historical range.

 

 

Histopathology

Microscopic findings considered to be treatment related were observed for the liver and kidneys.

 

For the liver, diffuse midzonal/centrilobular hypertrophy at minimal degree were recorded in 1/5 males at 250 mg/kg bw/day, and at minimal or slight degree for 6/7 males and at minimal degree in 1/5 females at 750/500 mg/kg bw/day.

 

For the kidneys, cortical hyaline droplets were increased in severity up to moderate degree in all treated groups of males. Tubular dilation with atrophic epithelium was recorded for males at up to moderate degree at 250 mg/kg bw/day and to very severe degree at 750/500 mg/kg bw/day and for females up to slight degree at 250 and at 750/500 mg/kg bw/day. Hyaline/granular casts up to moderate degree were present in 5/5 males at 250 mg/kg bw/day and up to severe degree in 8/10 males at 750/500 mg/kg bw/day and at slight degree in 1/5 females at 85 mg/kg bw/day, minimal in 2/5 females at 250 mg/kg bw/day and at minimal to moderate in 2/5 females at 750/500 mg/kg bw/day. Granulolymphocytic inflammatory infiltrate was increased in incidence and severity up to moderate for males at 250 mg/kg bw/day and 750/500 mg/kg bw/day and up to slight degree in females at 750/500 mg/kg bw/day. Corticomedullary tubular basophilia was increased in incidence and severity to severe in males and up to moderate in females at 250 and at 750/500 mg/kg bw/day. Papillary necrosis at slight or moderate degree was noted in the two decedents males at 750/500 mg/kg bw/day.

 

 

Conclusion

Nephrotoxicity at 250 mg/kg bw/day and at 750/500 mg/kg bw/day precluded these dosages from representing a No Observed Adverse Effect Level (NOAEL) for systemic toxicity therefore the NOAEL for systemic toxicity was considered to be 85 mg/kg bw/day.

 

As lower pregnancy rate was observed at 750/500 mg/kg bw/day, the clear No Observed Effect Level (NOEL) for reproduction was considered to be 250 mg/kg bw/day. The lower pregnancy rate at 750 mg/kg bw/day may have been a consequence of adverse adult toxicity apparent during the pre-pairing phase. Despite this adverse toxicity for the parental animals, there was no effect on their litters and the NOEL for the survival, growth and development of the offspring was at least 500 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

85 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In an Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) the NOAEL for systemic toxicity was found to be 85 mg/kg/day. Effects were noted at 250 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only this study is available and the study is Klimisch 1.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).