3-methyl-1-benzofuran-5-ol

Endpoint summary

Description of key information

The repeated dose toxicity of the test substance, TM 11-0230, was assessed according to OECD Test Guideline 422 using a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening method. The No Observed Effect Level (NOEL) for females was considered to be 2500 ppm (equivalent to a mean achieved dosage of 162.9 mg/kg bw/day) due to the microscopic changes evident in the oesophagus and stomach at 7500 ppm. A No Observed Effect Level (NOEL) for males was not established due to the presence of hyaline droplets in all treated males. However if this is ignored, considering it is male rat specific and not relevant to humans, the NOEL for males becomes 2500 ppm (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day). A “No Observed Adverse Effect Level” (NOAEL) can be established at 2500 ppm for animals of either sex (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day for males and 162.9 mg/kg bw/day for females), based on the observed effects.

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 12 November 2014 and 10 November 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 422 using a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening method and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for 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 302 to 363g, the females weighed 186 to 207g, 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 ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, 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 final week of 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 these targets.

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: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Test Item Preparation
For the purpose of this study the test item was incorporated into the diet at concentrations of 750, 2500 and 7500 ppm as follows:

A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.

The stability and uniformity of distribution of the test item in the diet were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results showed the dietary admixtures up to 15000 ppm to be stable for twenty-two days when stored at room temperature in the dark. The diet was stored in labelled, double plastic bags in labelled, covered plastic bins at room temperature.

Samples were taken from the dietary admixtures and analysed for uniformity of distribution and concentration at Harlan Laboratories Ltd, Shardlow, UK, Analytical Services. The results indicate that the mean prepared dietary admixture concentrations were within 94 to 100% of the nominal concentrations.

Procedure
The test item was administered continuously by dietary admixture. Control animals were treated in an identical manner with basal laboratory diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test samples was determined by gas chromatography (GC) 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 methanol were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was accurately weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1000 ppm. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 100 ppm. Standard solutions contained the equivalent amount of diet to that of the relevant samples.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The dietary admixtures received were extracted with methanol. An aliquot, approximately 5 g of dietary admixture was accurately weighed into a suitable jar and the correct amount of methanol added which was then ultra-sonicated for 15 minutes and left to extract overnight in ambient conditions. The sample solutions were then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, samples solutions were further diluted with methanol to achieve the working concentration.

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

Preparation of linearity standards
A range of standard solutions were prepared in methanol from a stock solution of 1008 ppm by serial dilution covering the concentration range 0 to 151.2 ppm.

Instrumental setup
GC system: Agilent technologies 5890, 6890 and 7890 incorporating autosampler and workstation
Column: ZB-5 (30 m x 0.53 mm if x 5 µm film).
Oven temperature program: 100 °C for 0 minutes with 10 °C / minute to 280 °C for 5 minutes
Injection temperature: 300 °C
Flame ionisation detector temperature: 300 °C
Injection volume: 1 µL
Retention time: ~ 10 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 method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention 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 with the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

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

Test item dietary admixtures
The admixtures investigated during the study were found to comprise test item in the range of 94 % to 100 % and thus the required content limit of ± 20 % with reference to the nominal content was met.
The test item was found to be stable in the admixtures when kept for 22 days at room temperature due to results which met the variation limit of 20 % from time-zero mean.
In conclusion, the results indicate the accurate use of the test item and diet 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:

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

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
750, 2500 and 7500 ppm (equivalent to a mean achieved dosage of 42.4, 138.0 and 412.8 mg/kg bw/day for males
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
56.1, 162.9 and 463.0 mg/kg bw/day for females during the pre-pairing phase
Basis:
nominal in diet

No. of animals per sex per dose:

Twelve males and twelve females

Control animals:

yes, plain diet

Details on study design:

Chronological Sequence of Study
i. Animals received treated diet, according to dose group throughout the study period. Control animals received basal laboratory diet.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. Vaginal smears were taken for all control and treated females during the pre-pairing phase.
iv. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v. 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.
vi. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vii. 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.
viii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix. 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.
x. 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.

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily. 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 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 and then weekly for males until termination and weekly for females until pairing. 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.


Estrous Cycle
A vaginal smear was taken daily for females during the pre-pairing period. The sample was placed on a glass slide and the smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of estrus was recorded.


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. 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 least twice (early morning and as late as possible during the normal working day) 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 a suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. 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 a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). 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 in bold were preserved from all remaining animals:
Ovaries, Pituitary, Prostate, Seminal vesicles, Coagulating gland, Epididymides, Gross lesions, Testes, Mammary gland, Uterus/Cervix, Vagina
* = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were dispatched to the Test Site (Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from five selected control and 7500 ppm dose group animals and any animals which did not 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 from the remaining control and 7500 ppm animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 7500 ppm 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.

Since there were indications of treatment-related stomach, esophagus and kidney (males only) changes, examination was subsequently extended to include similarly prepared sections of the stomach, esophagus and kidney (males only) from animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (R Alison at Roger Alison Ltd). A peer review of the histopathology examinations was also performed by W Henderson.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
There were no unscheduled deaths.

Clinical Observations
There were no treatment-related clinical signs apparent for animals of either sex treated with 750, 2500 or 7500 ppm.
One control female had a wound on the right side of the neck on Days 16 and 17, followed by a scab between Days 18 and 30. This was considered to be a physical injury that occurred during mating.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioral parameters at 750, 2500 or 7500 ppm.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.
Animals of either sex treated with 7500 ppm showed a statistically significant increase in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any associated changes to suggest a neurotoxic effect, the intergroup differences were considered not to be of toxicological importance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

Body Weight
Males treated with 7500 or 2500 ppm showed a statistically significant reduction in body weight gain during the first week of treatment. Body weight gain for males at 2500 ppm was similar to controls thereafter. Initial recovery was evident for males at 7500 ppm however; body weight gains during Week 4 was lower than controls, and attained statistical significance. Overall body weight gain for males at 7500 ppm was lower than controls.

Body weight gain for females at 7500 ppm was lower than controls during the first week of treatment and throughout gestation and lactation. Cumulative body weight gain during gestation was statistically significantly lower than controls at this dietary concentration and statistically significantly lower absolute body weights from Day 7 of gestation through to Day 4 of lactation was evident.

No such effects were detected for females treated with 2500 or animals of either sex treated with 750 ppm.

Food Consumption
At 7500 ppm, food consumption for males was slightly lower than controls throughout the study. Food consumption for females from this treatment group was comparable to controls during the pre-pairing phase however, reduced consumption was evident throughout gestation and lactation. Differences from controls attained statistical significance between Days 7 and 14 of gestation and during lactation. Instances of reduced food conversion efficiency was evident in animals of either sex treated with 7500 ppm during the first week of treatment.

No such effects were detected for animals of either sex treated with 2500 or 750 ppm.

Water Consumption
Daily gravimetric assessment of water bottles during the pre-pairing period did not reveal any significant intergroup differences.

Estrous Cycle Assessments
There were no treatment-related effects on female estrous cycles or on proportion of females with anomalous oestrous cycles.

Two females treated with 750 ppm, one female treated with 2500 ppm and one female treated with 7500 ppm showed extended di-estrus during the pre-pairing phase. All of these females showed positive evidence of mating however one of the 750 ppm females and the 2500 ppm female were non-pregnant. In the absence of a true dose related response or an effect on mating or fertility, the intergroup differences were considered to be incidental.

Reproductive Performance
Mating
There were no treatment-related effects on mating performance.

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

One female treated with 750 ppm and one female treated with 2500 ppm were not pregnant following positive evidence of mating. No histopathological abnormalities were observed in either the males or females to explain the failure of the animals to breed successfully and in the absence of a similar effect in the 7500 ppm dose group, the intergroup differences were considered to be incidental and of no toxicological significance.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 23½ days.

Litter Responses
In total all females from the control group and eleven females from the 750, 2500 and 7500 ppm dose groups gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 7500 ppm gave birth to a live litter but had a total litter loss prior to Day 5 post partum. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant differences were detected for corpora lutea, implantation counts or implantation losses for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

A slight reduction in litter size at birth and on Days 1 and 4 post partum was evident in females treated with 7500 ppm; however offspring survival to Day 5 of age was considered unaffected by treatment. A slight reduction in offspring viability was evident at 7500 ppm however this was considered to be the result of one litter which lost one offspring between Days 1 and 4 post partum. A loss of one offspring is not uncommon therefore this was considered of no toxicological significance. No such effects were detected at 2500 or 750 ppm.

There were no toxicologically significant differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

One female treated with 7500 ppm had a total litter loss on Day 3 post partum. This female only had one live offspring in the litter and had a slightly longer parturition length (23½ Days) which may have compromised offspring post-natal survival. In the absence of an effect on offspring survival in the remaining litters at this dose group, this total litter loss was considered unrelated to treatment.

Offspring Growth and Development
Marginally lower litter size was evident in females treated with 7500 ppm on Days 1 and 4 post partum. This resulted in lower litter weights on Days 1 and Day 4 post partum, with differences from controls achieving statistical significance on Day 4 post partum.

No such effects were detected at 2500 or 750 ppm.

Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected consisting of small size, weak, no milk in stomach, missing and found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Laboratory Investigations
Hematology
There were no toxicologically significant effects detected in the hematological parameters examined.

Animals of either sex treated with 7500 ppm showed a statistically significant increase in activated partial thromboplastin time. The majority of the individual values were within the normal background range and in the absence of any associated histopathological changes, the intergroup differences were considered of no toxicological importance. Males treated with 7500 and 2500 ppm showed statistically significant increases in total leukocyte count and neutrophil count and a statistically significant reduction in hemoglobin. The majority of the individual values were within the normal background range for these parameters and in the absence of any associated histopathology correlates or a true dose related response, the intergroup differences were considered of no toxicological importance.

Blood Chemistry
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Females treated with 7500 ppm showed statistically significant increases in bilirubin and chloride concentration. Males from this treated group also showed a statistically significant reduction in creatinine. All of the individual values were within the normal background ranges for these parameters and in the absence of any associated histopathological changes, the intergroup differences were considered of no toxicological importance.

Pathology
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.

Adults
No toxicologically significant macroscopic abnormalities were detected.

One control female, four females treated with 750 ppm and one male and two females treated with 7500 ppm had reddened lungs at necropsy. In the absence of any associated treatment-related histopathological correlates and in view of the fact that one control animal also showed the effect, the intergroup differences were considered to be incidental and of no toxicological significance.

One female treated with 750 ppm had increased pelvic space in the left kidney. This observation is considered to be a congenital abnormality and unrelated to treatment.

Organ Weights
Males treated with 7500 and 2500 ppm showed a statistically significant increase in absolute and body weight-relative kidney weight when compared to controls. Although a true dose related response was not evident, the increased weights did correlate with hyaline droplets seen microscopically in treated males.

No toxicologically significant effects were detected in females treated with 7500 or 2500 ppm or in animals of either sex treated with 750 ppm.

Females treated with 7500 and 2500 ppm showed a statistically significant increase in uterus weight both absolute and relative to terminal body weight. Females treated with 2500 ppm also showed a statistically significant reduction in brain weight both absolute and relative to terminal body weight. Females treated with 750 ppm showed a statistically significant increase in absolute and body weight relative spleen weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any associated histopathological correlates or a true dose related response, the intergroup differences were considered not to be of toxicological significance.

Males treated with 7500 ppm showed a statistically significant reduction in seminal vesicle weight both absolute and relative to terminal body weight. Males treated with 2500 ppm showed a statistically significant increase in pituitary weight both absolute and relative to terminal body weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Histopathology
The following treatment-related microscopic abnormalities were detected:

Kidneys: Hyaline droplets were evident in males from all treatment groups.

Oesophagus: Hyperkeratosis was evident in animals of either sex treated with 7500 ppm.

Stomach: Acanthosis and hyperkeratosis was evident in the fore stomach of animals of either sex treated with 7500 ppm.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Due to the microscopic changes evident in the oesophagus and stomach at 7500 ppm.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: 2500 ppm for animals of either sex

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: 2500 ppm for animals of either sex

Critical effects observed:

not specified

Achieved Intake

Group mean achieved dosage of IFF TM 11-0230 in mg/kg bw/day during the study is given, calculated using actual concentrations for dietary formulations.

 

At 7500 ppm, mean achieved dosage for males was 412.8 mg/kg bw/day. Mean achieved intakes were fairly consistent and generally maintained the intended ten fold interval between this high dietary level and the low dietary level. Mean achieved dosage for females during the pre-pairing period was 463.0 mg/kg bw/day. Average mean achieved intake was a 8.2 fold interval between this high dietary level and the low dietary level during the pre-pairing period.

 

At 2500 ppm, mean achieved dosage for males was 138.0 mg/kg bw/day and for females during the pre-pairing period was 162.9 mg/kg bw/day. Mean achieved intake was fairly consistent and generally maintained a 3.3 fold interval for males and a 2.9 fold interval for females between this intermediate dietary level and the low dietary level.

 

At 750 ppm, mean achieved dosage for males was 42.4 mg/kg bw/day and for females during the pre-pairing period was 56.1 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period.

Conclusions:

The oral administration of IFF TM 11-0230 to rats for a period of approximately six weeks (including two weeks pre-pairing, gestation and early lactation for females) at dietary concentrations of up to 7500 ppm, resulted in treatment related effects detected in animals of both sexes treated with 7500 ppm and in males treated with 2500 and 750 ppm. The No Observed Effect Level (NOEL) for females was considered to be 2500 ppm (equivalent to a mean achieved dosage of 162.9 mg/kg bw/day) due to the microscopic changes evident in the oesophagus and stomach at 7500 ppm. A No Observed Effect Level (NOEL) for males was not established due to the presence of hyaline droplets in all treated males. However if this is ignored considering it is male rat specific and not relevant to humans, the NOEL for males becomes 2500 ppm (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day).

The effect on body weight gain and food consumption/food conversion efficiency in both males and females at 7500 ppm and in males treated with 2500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment. The stomach and oesophagus changes identified at 7500 ppm may be considered to be an adverse effect of treatment, although they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. A “No Observed Adverse Effect Level” (NOAEL) can be established at 2500 ppm for animals of either sex (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day for males and 162.9 mg/kg bw/day for females), based on the observed effects excluding those related to alpha 2u-globulin nephropathy.

The ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 2500 ppm (equivalent to a mean achieved dosage of 162.9 mg/kg bw/day in females) due to lower litter weights on Days 1 and 4 post partum at 7500 ppm (with statistical significance achieved on Day 4 post partum), which was caused by marginally lower litter size. A relationship with reduced maternal body weight gain during gestation and lactation as a consequence of reduced food consumption due to gastric irritancy caused by the test substance in the diet cannot be excluded for the lower litter weights at 7500 ppm.

Executive summary:

The repeated dose toxicity of the test substance, TM 11-0230, was assessed according to OECD Test Guideline 422 using a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening method. The No Observed Effect Level (NOEL) for females was considered to be 2500 ppm (equivalent to a mean achieved dosage of 162.9 mg/kg bw/day) due to the microscopic changes evident in the oesophagus and stomach at 7500 ppm. A No Observed Effect Level (NOEL) for males was not established due to the presence of hyaline droplets in all treated males. However if this is ignored, considering it is male rat specific and not relevant to humans, the NOEL for males becomes 2500 ppm (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day). A “No Observed Adverse Effect Level” (NOAEL) can be established at 2500 ppm for animals of either sex (equivalent to a mean achieved dosage of 138.0 mg/kg bw/day for males and 162.9 mg/kg bw/day for females), based on the observed effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

138 mg/kg bw/day

Study duration:

subacute

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

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

Specific organ toxicity (repeated exposure) is defined as specific target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed, are included. Classification for target organ toxicity (repeated exposure) identifies the substance as being a specific target organ toxicant and, as such, it may present potential for adverse health effects in people who are exposed to it. These adverse health effects include consistent and identifiable toxic effects in humans, or in experimental animals toxicologically significant changes which have affected the function or morphology of a tissue or organ, or have produced serious changes to the biochemistry or haematology of the organism and these changes are relevant for human health. Assessment takes into consideration not only significant changes in a single organ or biological system but also generalised changes of a less severe nature involving several organs.

 

Substances are classified in two categories:

Category 1 includes substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure. Substances are classified in Category 1 for target organ toxicity (repeat exposure) on the basis of reliable and good quality evidence from human cases or epidemiological studies; or observations from appropriate studies in experimental animals in which significant and / or severe toxic effects of relevance to human health were produced at generally low exposure concentrations. For a 90-Day repeated dose oral study performed in rats, a broad guidance value for Category 1 via the oral route is ≤ 10 mg/kg body weight/day. This guidance value is increased by a factor of 3 for 28-day studies.

 

Category 2 includes substances that, on the basis of studies in experimental animals, can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in Category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects of relevance to human health were produced at generally moderate exposure concentrations. For a repeated dose oral study performed in rats, the broad guidance value for Category 2 via the oral route is ≤ 100 mg/kg body weight/day. This guidance value is increased by a factor of 3 for 28-day studies.

During the study, males treated with 7500 ppm (equivalent to 412.8 mg/kg bw) had reduced body weight development with a reduction in overall bodyweight gain for these males, along with a reduction in overall food consumption for males at this dosage and reduction in food efficiency. Similarly, males treated with 2500 ppm (equivalent to 138 mg/kg bw) also showed a reduction in body weight gain, however recovery was evident. Females treated with 7500 ppm (equivalent to 463 mg/kg bw) had reduced body weight, a reduction in body weight gain and food consumption throughout gestation and lactation and a reduction in food conversion efficiency.

Reduced body weight gains and food consumption are often reported when the dietary admixture is unpalatable or irritant and can be associated with gastric irritancy rather than attributable to systemic toxicity. This was supported microscopically, with stomach changes identified as hyperkeratosis and acanthosis of the fore stomach in animals of either sex treated with 7500 ppm and hyperkeratosis in the oesophagus of these animals. This indicates that while these findings represent an adverse effect of treatment, they are considered to reflect local irritation rather than any adverse systemic toxicity of the test item.

Although there were some statistically significant differences in treated animals from controls for the hematological and blood chemical parameters measured, these differences were considered not to be of toxicological significance due to the majority of individual values being within normal background ranges and no associated histopathological correlates.

Kidney weights were elevated in males treated with 7500 and 2500 ppm and there was an increase in incidence of hyaline droplets in the kidneys of males from all treatment groups. Hyaline droplets can be directly linked to the accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is not found in immature rats, female rats or humans and therefore is considered to be of no toxicological relevance to man.

Given the nature of these effects, the test substance is not classified for specific organ toxicity (repeated dose).