3-(p-methoxyphenyl)-2-methylpropionaldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Oct 2016 to 30 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Oct 2016 to 30 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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)

Version / remarks:

adopted 22 March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 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).

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Males: Appr. 11 weeks, Females Appr. 14 weeks
- Weight at study initiation: Males: 283g to 349g, Females: 182g to 233g
- Fasting period before study: no
- Housing: 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. 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.
- Diet: A ground or powdered diet (Rodent PMI 5002 (Certified) Ground Diet (BCM IPS Limited, London, UK)) was provided ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
- Acclimation period: Eighteen days during which time health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Mar 2017 to 16 May 2017

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): 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/U200 mixer.
- Storage temperature of food: Room temperature, with the exception of the final mix which was prepared for approximately two weeks and split into two weekly aliquots. When in use, the first aliquot was stored at room temperature and the second aliquot, when not in use was kept in labelled, double plastic bags and stored in the freezer (approximately -20 °C).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: 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)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eight days at room temperature and twenty-one days at approximately -20 °C.
- Samples were taken from the dietary admixtures on four occasions and analyzed for uniformity of distribution and concentration of the substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 19% of the nominal concentration.
- The analytical method was based on GC using FID detection

Duration of treatment / exposure:

six weeks (males) or up to eight weeks (females, including a two week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Continuous by dietary admixture

Dose / conc.:

600 ppm

Remarks:

Mean achieved dosage levels: 39.6 mg/kg bw/day (males), 47.1 mg/kg bw/day (females pre-mating), 52.5 mg/kg bw/day (females during gestation) and 96.2 mg/kg bw/day (females during lactation)

Dose / conc.:

2 000 ppm

Remarks:

Mean achieved dosage levels: 129.7 mg/kg bw/day (males), 161.4 mg/kg bw/day (females pre-mating), 174.1 mg/kg bw/day (females during gestation) and 289.5 mg/kg bw/day (females during lactation)

Dose / conc.:

6 000 ppm

Remarks:

Mean achieved dosage levels: 389.9 mg/kg bw/day (males), 451.3 mg/kg bw/day (females pre-mating), 553.2 mg/kg bw/day (females during gestation) and 859.8 mg/kg bw/day (females during lactation)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Basis for dose level selection: The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work (including a 14 Day range-finding toxicity study in the rat.
- The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily. All observations were recorded.
- 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: date of pairing, date of mating, date and time of observed start of parturition, date and time of observed completion of parturition.

DETAILED CLINICAL OBSERVATIONS:
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 and Lachrymation.

BODY WEIGHT:
Individual body weights were recorded on Day 1 and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND FOOD EFFICIENCY
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 for the periods covering Days 1-4, 4-7 and 7-14 post partum.
Food efficiency (the ratio of body weight change/dietary intake) and mean achieved dosages were calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation. Mean achieved dosages for females during gestation and lactation have been calculated, however, these values may have been affected by offspring growth during gestation and the possibility of offspring starting to eat the diet during the final week of treatment.

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

HAEMATOLOGY
- Haematological investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 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.
- The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT) and Reticulocyte count (Retic).
- 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).

CLINICAL CHEMISTRY
- Blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 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.
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids and Calcium (Ca++)

THYROID HORMONE ANALYSIS
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into potassium EDTA tubes, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
- Serum and plasma samples were taken from all adult males and females at termination.
- The serum from adult males was analyzed for Thyroxine (T4)

NEUROBEHAVIOURAL EXAMINATION: Functional observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. 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.

NEUROBEHAVIOURAL EXAMINATION: 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.
- 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.

SENSORY REACTIVITY
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex and Finger approach

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

During histopathology 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.

Litter observations:

PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
- Sex of offspring on Days 1, 4, 7 and 13 post partum
- Clinical condition of offspring from birth to Day 13 post partum
- Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
- All live offspring were assessed for ano-genital distance on Day 1 post partum.
- Visible nipple count was performed for all male offspring on Day 13 post partum.

THYROID HORMONE ANALYSIS
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into potassium EDTA tubes, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
- Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring are of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
- Serum samples from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where required the number/sex of offspring sampled was altered depending on the litter constituents.
- The serum from Day 13 offspring was analyzed for Thyroxine (T4)

Postmortem examinations (parental animals):

SACRIFICE
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed around the same time as littering females.

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

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Brain, Spleen, Heart, Kidneys, Thymus, Liver
- From all remaining animals the following organs where weighted: Pituitary (post-fixation), Prostate and Seminal Vesicles (with Coagulating Gland), Epididymides, Testes, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix and oviducts)

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 otherwise stated: Adrenals, Aorta (thoracic), Muscle (skeletal), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Pancreas, Brain (including cerebrum, cerebellum and pons), Caecum, Rectum, Colon, Salivary glands (submaxillary), Cowpers Glands, Sciatic nerve, Duodenum, Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (in Davidsons fluid), Glans Penis, Spleen, Stomach, Heart, Ileum (including Peyer’s patches), Jejunum, Trachea, Kidneys, Thymus, LABC (levator ani-bulbocavernous) muscle, Urinary bladder, Liver, Lungs (with bronchi, lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative) and Lymph nodes (mandibular and mesenteric).
- For remaining animals the following organs were preserved: Mammary gland, Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides (in Modified Davidsons fluid), Gross lesions, Testes (in Modified Davidsons fluid), Thyroid/Parathyroid, Uterus & Cervix and Vagina.
- The tissues from five selected control and 6000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues as indicated from the remaining control and 6000 ppm animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 6000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
- Immunohistochemical staining of the kidneys for α-2u-globulin was also performed (from additional sections from both kidneys) for five selected males from the control and high dose groups.
- Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the spleen from five selected females in the low and intermediate groups.

OTHER
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

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS NECROPSY
All offspring, including those dying during the study, were subjected to an examination. Examination of offspring was restricted to an macroscopic external examination, except where abnormalities were observed an additional internal examination was performed.

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'

Reproductive indices:

- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Mating Index (%) = (Number of animals mated/ Number of animals paired) x 100
- Pregnancy index (%) = (Number of pregnant females/ Number of animals mated) x 100
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index (%) = (Number of females delivering live offspring/ Number of pregnant females) x 100

Offspring viability indices:

- Post–implantation loss (%) = (Number of implantation sites - Total number of offspring born/ Number of implantation sites) x 100
- Live Birth Index (%) = (Number of offspring alive on Day 1/ Number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 13/ Number of offspring alive on Day 4) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent for animals of either sex fed diet containing 600, 2000 or 6000 ppm.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males fed diet containing 6000 ppm showed a reduction in body weight gain from Week 3 onwards. Statistical significance was achieved during Weeks 5 (p<0.01) and 6 (p<0.05). Overall body weight gain for these males was 23% lower than controls.
- Females from all dietary groups showed a slight reduction in body weight gain during maturation; however, statistical significance was not achieved and a true dose related response was also not evident, therefore the intergroup differences were considered of no toxicological significance. Body weight gain in all treated females during gestation and lactation was comparable to controls.
- A slight reduction in body weight gain was evident in males fed diet containing 2000 and 600 ppm during Week 3. Statistical significance was not achieved and recovery was evident during Week 4. A statistically significant reduction (p<0.05) in body weight gain was evident for these males during Week 5, however, statistical significance was minimal and a true dose related response was not evident. The intergroup differences evident at 2000 and 600 ppm were therefore considered not to represent a true treatment-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- No adverse effect on food consumption was evident in treated males.
- No adverse effect on food consumption was evident in treated females during maturation or gestation. Females fed diet containing 6000 ppm showed a statistically significant reduction (p<0.001) in food consumption between Days 4 and 7 of lactation. A reduction in food consumption continued for the remainder of lactation (Days 7-14), however, statistical significance was not achieved.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Minor fluctuations in food conversion efficiency were evident in males fed diet containing 6000 ppm during Weeks 5 and 6, however, these were considered to reflect the changes in body weight gain seen in these males and were considered not to be of toxicological significance.

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the haematology parameters examined. Animals of either sex from all dietary concentrations showed statistically significant increases (p<0.01) in reticulocyte count. All of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
- Males fed diet containing 6000 ppm showed statistically significant reductions in alanine aminotransferase (p<0.05) and cholesterol (p<0.01) and a statistically significant increase in bilirubin (p<0.05). The majority of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
- Females from all dietary concentrations showed a statistically significant increase in chloride concentration (p<0.01) and a statistically significant reduction in cholesterol (p<0.05-0.01). With the exception of two cholesterol values (one female fed diet containing 2000 ppm and one female fed diet containing 6000 ppm), all of the remaining individual values for cholesterol and chloride concentration were within background control ranges. A true dose related response was also not evident for chloride concentration and no associated histopathological correlates were evident. The intergroup differences were therefore considered not to be of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment-related changes in the behavioral parameters at 600, 2000 or 6000 ppm.
- Sensory reactivity scores across all test item-treated dose groups were similar to controls.
- Functional performance tests: There were no intergroup differences at any dietary concentration considered to be related to treatment with the test item. Males fed diet containing 6000 ppm showed a statistically significant increase in hind limb grip strength (p<0.05-0.01) in two out of the three tests. During motor activity evaluations, males from this dietary group also showed a statistically significant reduction (p<0.05) in the final 20% of activity monitoring time and females showed statistically significant increases (p<0.01) in overall activity and the final 20% of activity monitoring time. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The following treatment-related microscopic abnormality was detected in the spleen: A reduction in hematopoiesis was evident in females fed diet containing 6000 ppm. All females from the control, 600 and 2000 ppm groups had minimal or mild hematopoiesis as generally seen in lactating females in this type of study. Hematopoiesis was not recorded in any females fed diet containing 6000 ppm. No such effect was evident treated males.
- Special stains of male kidney sections, for the presence of α-2u-globulin were examined and did not indicate significant or consistent differences between control and males fed diet containing 6000 ppm.
- There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item at any dietary concentration on the nature of estrous cycle, with the majority of females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- There was no adverse effect of treatment on mating performance. With the exception of one control female, two females fed diet containing 2000 ppm and two females fed diet containing 6000 ppm, all animals mated within the first five days after pairing. These remaining females mated within the fourteen day pairing period.
- Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level. One female fed diet containing 6000 ppm and two females fed diet containing 2000 ppm were found to be non-pregnant following positive evidence of mating. Histopathological examinations of the female and the respective male partners at 2000 ppm did not reveal any significant microscopic changes which could account for the lack of pregnancy therefore these were considered incidental and unrelated to treatment. The male partner to the nonpregnant female at 6000 ppm, had tubular atrophy in the testes, with subsequent aspermia in the epididymis. This was considered most likely to have resulted in the infertility observed. In isolation, this was considered incidental and unrelated to treatment.
- Gestation lengths were between 22 and 23½ days and the distribution for treated females was essentially similar to control.
- There was no effect of treatment with the test item at any dietary concentration on the mean number of implantations and post-implantation losses.
- In total all females from the control and 600 ppm dietary groups, ten females from the 2000 ppm dietary group and eleven females from the 6000 ppm dietary group gave birth to a live litter and successfully reared young to Day 13 of age.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

129.7 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

161.4 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

389.9 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Highest dose tested

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

451.3 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs detected in pups from treated dose groups included small size, pale, cold, no milk in stomach, physical injury and found dead or missing. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Offspring body weight and litter weight on Day 1 post partum was comparable to controls.
- Offspring body weight, offspring body weight gain and litter weights from females fed diet containing 6000 ppm was reduced on Days 4, 7 and 13 post partum when compared to control litters. Statistical significance (p<0.05-0.001) was achieved for litter weight and offspring body weight on Days 7 and 13 and for offspring body weight gains/cumulative gains throughout lactation.
- No such effects were evident in litters from females fed diet containing 2000 or 600 ppm.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 600, 2000 and 6000 ppm.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings. Statistical analysis of the data did reveal a statistically significant reduction (p<0.01) in T4 values in female offspring from adult females litters fed diet containing 6000 ppm. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance.
- There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

289.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Achieved Intake

Group mean achieved dosage of substance in mg/kg bw/day during the study are calculated using nominal concentration for dietary formulations.

- At 6000 ppm, mean achieved dosage for males was 389.9 mg/kg bw/day and for females during the pre-pairing period was 451.3 mg/kg bw/day. Mean achieved intakes were fairly consistent for animals of either sex and generally maintained a 10 fold interval between this high dietary level and the low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 2000 ppm, mean achieved dosage for males was 129.7 mg/kg bw/day and for females during the pre-pairing period was 161.4 mg/kg bw/day. Mean achieved intake was fairly consistent for animals of either sex and generally maintained a 3 fold interval between this intermediate dietary level and low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 600 ppm, mean achieved dosage for males was 39.6 mg/kg bw/day and for females during the pre-pairing period was 47.1 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period and increased slightly during gestation and lactation due to the increased demand on the adult female.

Conclusions:

For the substance a parental NOAEL for systemic effects of 2000 ppm was derived based on food efficiency and body weight gain in males and body weight gain, food consumption, reduced spleen weight and reduction in the amount of haematopoiesis in the spleen of high dose females. The lowest derived NOAEL is 129.7 mg/kg bw/day based on the lower food intake in males. No reproduction toxicity was observed up to and including 6000 ppm, the highest dose level tested.

Executive summary:

A study was performed according to OECD TG 422 and GLP principles.The test item was administered by continuous dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 600, 2000 and 6000 ppm (equivalent to a mean achieved dosage of 39.6, 129.7 and 389.9 mg/kg bw/day for males and 47.1, 161.4 and 451.3 mg/kg bw/day respectively for females during maturation, 52.2, 174.1 and 553.2 mg/kg bw/day respectively for females during gestation and 96.2, 289.5 and 859.8 mg/kg bw/day respectively for females during lactation). A control group of twelve males and twelve females were treated with basal laboratory diet.

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 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only). Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4). Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Parental toxicity

No mortality or clinical signs were detected. In addition no changes in behavioral parameters, functional performance or sensory activity were observed. Regarding the body weight,males fed diet containing 6000 ppm showed a reduction in body weight gain from Week 3 onwards. Overall body weight gain for these males was reduced when compared to controls. No toxicologically significant effects were evident in males fed diet containing 2000 or 600 ppm. No toxicologically significant effects in body weight gain were evident in treated females during maturation. Body weight gain in all treated females during gestation and lactation was comparable to controls. No adverse effect on food consumption was evident in treated males. Food conversion efficiency was reduced in males fed diet containing 6000 ppm during Weeks 5 and 6 of treatment up to 44% at the end of exposure. No such effect was evident in males fed diet containing 2000 or 600 ppm. No adverse effect on food consumption or food conversion efficiency (maturation only) was evident in treated females during maturation or gestation; however, females fed diet containing 6000 ppm showed a reduction in food consumption between Days 4 and 14 of lactation. No such effect was evident in females fed diet containing 2000 or 600 ppm. Food efficiency (during maturation) was also reduced up to 19%. Daily visual assessment of water consumption did not reveal any significant intergroup differences. There were no toxicologically significant effects detected in the hematological parameters or blood chemistry examined. Upon Necropsy neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect up to a dietary concentration of 6000 ppm. Females fed diet containing 6000 ppm showed a statistically significant reduction in spleen weight both absolute and relative to terminal body weight. No such effects were detected in males fed diet containing 6000 ppm or animals of either sex fed diet containing 2000 or 600 ppm. A reduction in the amount of hematopoiesis in the spleen of females fed diet containing 6000 ppm was evident when compared to controls. No such effect was evident in any treated male or in females fed diet containing 2000 or 600 ppm. An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Fertility

Regarding the reproductive parameters, there was no effect of treatment with the test item at any dietary concentration on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy. There was no effect of treatment on mating performance. With the exception of five pairings, all animals mated within five days of pairing. There were no treatment-related effects in conception rates for test item-treated animals in relation to controls and no differences in gestation lengths in animals receiving the test item when compared with controls were observed.

Offspring

There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 600, 2000 and 6000 ppm. Offspring body weight and litter weight on Day 1post partumwas comparable to controls. Offspring body weight, offspring body weight gain and litter weights from females fed diet containing 6000 ppm was reduced on Days 4, 7 and 13 post partum when compared to control litters. No such effects were evident in litters from females fed 2000 or 600 ppm. There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13postpartumat 600, 2000 or 6000 ppm. An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.

 

For systemic toxicity some effects were seen on body weight (gain), food consumption and food efficiency at the high dose. In absence of clear palatability effects these effects are considered adverse at the high dose of 6000 ppm, resulting in a NOAEL of 2000 ppm (129.7 mg/kg bw/day for males and 161.4 mg/kg bw/day for females). The other effects seen in haematology, spleen and in adrenals are mild at the high dose and the relevance is questionable but these are also covered by this NOAEL. There were no effects on fertility, the ‘No Observed Adverse Effect Level’ (NOAEL) for fertility is considered to be ≥ 6000 ppm (389.9 mg/kg bw/day, based on the lower intake on males). The NOAEL for development toxicity is 2000 ppm (depending on the substance intake during lactation 289.5 mg/kg bw/day) based on reduced litter weights and reduced offspring body weight/body weight gains at 6000 ppm, which can be related to the lower food consumption and food efficiency in the dams.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Oct 2016 to 30 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 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).

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Males: Appr. 11 weeks, Females Appr. 14 weeks
- Weight at study initiation: Males: 283g to 349g, Females: 182g to 233g
- Fasting period before study: no
- Housing: 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. 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.
- Diet: A ground or powdered diet (Rodent PMI 5002 (Certified) Ground Diet (BCM IPS Limited, London, UK)) was provided ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
- Acclimation period: Eighteen days during which time health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Mar 2017 to 16 May 2017

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): 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/U200 mixer.
- Storage temperature of food: Room temperature, with the exception of the final mix which was prepared for approximately two weeks and split into two weekly aliquots. When in use, the first aliquot was stored at room temperature and the second aliquot, when not in use was kept in labelled, double plastic bags and stored in the freezer (approximately -20 °C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eight days at room temperature and twenty-one days at approximately -20 °C.
- Samples were taken from the dietary admixtures on four occasions and analyzed for uniformity of distribution and concentration of the substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 19% of the nominal concentration.
- The analytical method was based on GC using FID detection

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: 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)

Duration of treatment / exposure:

six weeks (males) or up to eight weeks (females, including a two week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Continuous by dietary admixture

Duration of test:

Females were dosed up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation) and were terminated together with surviving offspring on day 13 and 14 post partum

Dose / conc.:

600 ppm

Remarks:

Mean achieved dosage levels: 39.6 mg/kg bw/day (males), 47.1 mg/kg bw/day (females pre-mating), 52.5 mg/kg bw/day (females during gestation) and 96.2 mg/kg bw/day (females during lactation)

Dose / conc.:

2 000 ppm

Remarks:

Mean achieved dosage levels: 129.7 mg/kg bw/day (males), 161.4 mg/kg bw/day (females pre-mating), 174.1 mg/kg bw/day (females during gestation) and 289.5 mg/kg bw/day (females during lactation)

Dose / conc.:

6 000 ppm

Remarks:

Mean achieved dosage levels: 389.9 mg/kg bw/day (males), 451.3 mg/kg bw/day (females pre-mating), 553.2 mg/kg bw/day (females during gestation) and 859.8 mg/kg bw/day (females during lactation)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Basis for dose level selection: The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work (including a 14 Day range-finding toxicity study in the rat.
- The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily. All observations were recorded.
- 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: date of pairing, date of mating, date and time of observed start of parturition, date and time of observed completion of parturition.

DETAILED CLINICAL OBSERVATIONS:
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 and Lachrymation.

BODY WEIGHT:
Individual body weights were recorded on Day 1 and then weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND FOOD EFFICIENCY
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. 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 for the periods covering Days 1-4, 4-7 and 7-14 post partum.
Food efficiency (the ratio of body weight change/dietary intake) and mean achieved dosages were calculated retrospectively for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation. Mean achieved dosages for females during gestation and lactation have been calculated, however, these values may have been affected by offspring growth during gestation and the possibility of offspring starting to eat the diet during the final week of treatment.

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

HAEMATOLOGY
- Haematological investigations were performed on five females selected from each test and control group prior to termination (Day 13 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.
- The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT) and Reticulocyte count (Retic).
- 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).

CLINICAL CHEMISTRY
- Blood chemical investigations were performed on five females selected from each test and control group prior to termination (Day 13 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.
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids and Calcium (Ca++)

THYROID HORMONE ANALYSIS
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into potassium EDTA tubes, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
- Serum and plasma samples were taken from all adult females at termination.

NEUROBEHAVIOURAL EXAMINATION: Functional observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

NEUROBEHAVIOURAL EXAMINATION: 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.
- 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.

SENSORY REACTIVITY
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex and Finger approach

POST-MORTEM EXAMINATIONS
- Sacrifice: Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed around the same time as littering females.
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- The following organs were dissected free from fat and weighed before fixation from five selected females from each dose group: Adrenals, Brain, Spleen, Heart, Kidneys, Thymus, Liver. From all remaining animals the following organs where weighted: Pituitary (post-fixation), Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix and oviducts)

HISTOPATHOLOGY
- Samples of the following tissues were removed from five selected females from each dose group and preserved in buffered 10% formalin, except where otherwise stated: Adrenals, Aorta (thoracic), Muscle (skeletal), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Pancreas, Brain (including cerebrum, cerebellum and pons), Caecum, Rectum, Colon, Salivary glands (submaxillary), Cowpers Glands, Sciatic nerve, Duodenum, Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (in Davidsons fluid), Glans Penis, Spleen, Stomach, Heart, Ileum (including Peyer’s patches), Jejunum, Trachea, Kidneys, Thymus, LABC (levator ani-bulbocavernous) muscle, Urinary bladder, Liver, Lungs (with bronchi, lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative) and Lymph nodes (mandibular and mesenteric).
- For remaining animals the following organs were preserved: Mammary gland, Ovaries, Pituitary, Gross lesions, Thyroid/Parathyroid, Uterus & Cervix and Vagina.
- The tissues from five selected control and 6000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues as indicated from the remaining control and 6000 ppm animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 6000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
- Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the spleen from five selected females in the low and intermediate groups.

Ovaries and uterine content:

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. In addition, during histopathological examinations folicles and corpora lutea in the ovaries were examined.

Fetal examinations:

PARAMETERS EXAMINED
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
- Sex of offspring on Days 1, 4, 7 and 13 post partum
- Clinical condition of offspring from birth to Day 13 post partum
- Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
- All live offspring were assessed for ano-genital distance on Day 1 post partum.
- Visible nipple count was performed for all male offspring on Day 13 post partum.

SACRIFICE
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were killed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.

GROSS EXAMINATION OF DEAD PUPS:
All offspring, including those dying during the study, were subjected to an examination. Examination of offspring was restricted to an macroscopic external examination, except where abnormalities were observed an additional internal examination was performed.

THYROID HORMONE ANALYSIS
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into potassium EDTA tubes, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
- Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring are of the same sex, samples from the same litter were pooled). If eight or fewer offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
- Serum samples from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where required the number/sex of offspring sampled was altered depending on the litter constituents.
- The serum from Day 13 offspring was analyzed for Thyroxine (T4)

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'

Indices:

- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Mating Index (%) = (Number of animals mated/ Number of animals paired) x 100
- Pregnancy index (%) = (Number of pregnant females/ Number of animals mated) x 100
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index (%) = (Number of females delivering live offspring/ Number of pregnant females) x 100
- Post–implantation loss (%) = (Number of implantation sites - Total number of offspring born/ Number of implantation sites) x 100
- Live Birth Index (%) = (Number of offspring alive on Day 1/ Number of offspring born) x 100
- Viability Index 1 (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100
- Viability Index 2 (%) = (Number of offspring alive on Day 13/ Number of offspring alive on Day 4) x 100
- Sex Ratio (% males) = (Number of male offspring/Total number off offspring) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent for animalls fed diet containing 600, 2000 or 6000 ppm.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Females from all dietary groups showed a slight reduction in body weight gain during maturation; however, statistical significance was not achieved and a true dose related response was also not evident, therefore the intergroup differences were considered of no toxicological significance. Body weight gain in all treated females during gestation and lactation was comparable to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No adverse effect on food consumption was evident in treated females during maturation or gestation. Females fed diet containing 6000 ppm showed a statistically significant reduction (p<0.001) in food consumption between Days 4 and 7 of lactation. A reduction in food consumption continued for the remainder of lactation (Days 7-14), however, statistical significance was not achieved.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the haematology parameters examined. Animals from all dietary concentrations showed statistically significant increases (p<0.01) in reticulocyte count. All of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
- Females from all dietary concentrations showed a statistically significant increase in chloride concentration (p<0.01) and a statistically significant reduction in cholesterol (p<0.05-0.01). With the exception of two cholesterol values (one female fed diet containing 2000 ppm and one female fed diet containing 6000 ppm), all of the remaining individual values for cholesterol and chloride concentration were within background control ranges. A true dose related response was also not evident for chloride concentration and no associated histopathological correlates were evident. The intergroup differences were therefore considered not to be of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- There were no treatment-related changes in the behavioral parameters at 600, 2000 or 6000 ppm.
- Sensory reactivity scores across all test item-treated dose groups were similar to controls.
- Functional performance tests: There were no intergroup differences at any dietary concentration considered to be related to treatment with the test item. During motor activity evaluations, females from the 6000 ppm group showed statistically significant increases (p<0.01) in overall activity and the final 20% of activity monitoring time. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Females fed diet containing 6000 ppm showed a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. Although all of the individual values were within background control ranges, this reduction in weight does correlate with the microscopic changes evident in the spleen of these animals. Therefore, a relationship to treatment cannot be excluded.
- No such effects were detected in animals fed diet containing 2000 or 600 ppm.
- Females fed diet containing 2000 and 6000 ppm showed a statistically significant increase (p<0.05) in adrenal weights both absolute and relative to terminal body weight. All of the individual values were within background control ranges and in the absence of a true dose-related response, the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic findings detected in adult animals. Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included a mottled liver (one control female and one 2000 ppm female) and a pale liver (one 600 ppm female and one 2000 ppm female).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The following treatment-related microscopic abnormality was detected in the spleen: A reduction in hematopoiesis was evident in females fed diet containing 6000 ppm. All females from the control, 600 and 2000 ppm groups had minimal or mild hematopoiesis as generally seen in lactating females in this type of study. Hematopoiesis was not recorded in any females fed diet containing 6000 ppm.
- There were no test item-related microscopic findings in the reproductive tracts following the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no detrimental effect of treatment with the test item on the mean number of implantations or post-implantation loss at 600, 2000 and 6000 ppm.

Total litter losses by resorption:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no differences in gestation lengths in animals receiving the test item when compared with controls.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect of treatment on mating performance. With the exception of five pairings, all animals mated within five days of pairing.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related microscopic findings in the reproductive tracts follwing the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Key result

Dose descriptor:

NOAEL

Effect level:

161.4 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Offspring body weight and litter weight on Day 1 post partum was comparable to controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Offspring body weight, offspring body weight gain and litter weights from females fed diet containing 6000 ppm was reduced on Days 4, 7 and 13 post partum when compared to control litters. Statistical significance (p<0.05-0.001) was achieved for litter weight and offspring body weight on Days 7 and 13 and for offspring body weight gains/cumulative gains throughout lactation. No such effects were evident in litters from females fed diet containing 2000 or 600 ppm.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no detrimental effect of treatment with the test item on litter size at 600, 2000 and 6000 ppm.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.

External malformations:

no effects observed

Description (incidence and severity):

Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 600, 2000 and 6000 ppm.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.
- An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings. Statistical analysis of the data did reveal a statistically significant reduction (p<0.01) in T4 values in female offspring from adult females litters fed diet containing 6000 ppm. However, in the absence of a similar effect in male offspring or adult males the intergroup difference was considered not to be of toxicological significance.

Key result

Dose descriptor:

NOAEL

Remarks:

development

Effect level:

289.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

859.8 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Achieved Intake

Group mean achieved dosage of substance in mg/kg bw/day during the study are calculated using nominal concentration for dietary formulations.

- At 6000 ppm, mean achieved dosage for males was 389.9 mg/kg bw/day and for females during the pre-pairing period was 451.3 mg/kg bw/day. Mean achieved intakes were fairly consistent for animals of either sex and generally maintained a 10 fold interval between this high dietary level and the low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 2000 ppm, mean achieved dosage for males was 129.7 mg/kg bw/day and for females during the pre-pairing period was 161.4 mg/kg bw/day. Mean achieved intake was fairly consistent for animals of either sex and generally maintained a 3 fold interval between this intermediate dietary level and low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 600 ppm, mean achieved dosage for males was 39.6 mg/kg bw/day and for females during the pre-pairing period was 47.1 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period and increased slightly during gestation and lactation due to the increased demand on the adult female.

Conclusions:

Based on the findings in this study, the NOAEL for maternal and developmental toxicity was 2000 and 6000 ppm (corresponding to 161.4 and 289.5 mg/ kg bw/day) respectively. The NOAEL for developmental toxicity was based on reduced pub body weight (development) as a secondary effect to reduced maternal food intake.

Executive summary:

A study was performed according to OECD TG 422 and GLP principles.The test item was administered by continuous dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 600, 2000 and 6000 ppm (equivalent to a mean achieved dosage of 47.1, 161.4 and 451.3 mg/kg bw/day respectively for females during maturation, 52.2, 174.1 and 553.2 mg/kg bw/day respectively for females during gestation and 96.2, 289.5 and 859.8 mg/kg bw/day respectively for females during lactation). A control group of twelve males and twelve females were treated with basal laboratory diet.

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 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only). Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis. Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Maternal toxicity

No mortality or clinical signs were detected. In addition no changes in behavioral parameters, functional performance or sensory activity were observed. No toxicologically significant effects in body weight gain were evident in treated females during maturation. Body weight gain in all treated females during gestation and lactation was comparable to controls. No adverse effect on food consumption or food conversion efficiency (maturation only) was evident in treated females during maturation or gestation; however, females fed diet containing 6000 ppm showed a reduction in food consumption between Days 4 and 14 of lactation. No such effect was evident in females fed diet containing 2000 or 600 ppm. Food efficiency (during maturation) was also reduced up to 19%. Daily visual assessment of water consumption did not reveal any significant intergroup differences. There were no toxicologically significant effects detected in the hematological parameters or blood chemistry examined. Upon Necropsy neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect up to a dietary concentration of 6000 ppm. Females fed diet containing 6000 ppm showed a statistically significant reduction in spleen weight both absolute and relative to terminal body weight. No such effects were detected in females fed diet containing 2000 or 600 ppm. A reduction in the amount of hematopoiesis in the spleen of females fed diet containing 6000 ppm was evident when compared to controls. No such effect was evident in any females fed diet containing 2000 or 600 ppm.

Fertility

Regarding the reproductive parameters, there was no effect of treatment with the test item at any dietary concentration on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy. There was no effect of treatment on mating performance. With the exception of five pairings, all animals mated within five days of pairing. There were no treatment-related effects in conception rates for test item-treated animals in relation to controls and no differences in gestation lengths in animals receiving the test item when compared with controls were observed.

Offspring

There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 600, 2000 and 6000 ppm. Offspring body weight and litter weight on Day 1post partumwas comparable to controls. Offspring body weight, offspring body weight gain and litter weights from females fed diet containing 6000 ppm was reduced on Days 4, 7 and 13post partumwhen compared to control litters. No such effects were evident in litters from females fed 2000 or 600 ppm. There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13postpartumat 600, 2000 or 6000 ppm. An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.

 

For systemic toxicity some effects were seen on body weight (gain), food consumption and food efficiency at the high dose. In absence of clear palatability effects these effects are considered adverse at the high dose of 6000 ppm, resulting in a NOAEL of 2000 ppm (161.4 mg/kg bw/day). The other effects seen in hematology, spleen and in adrenals are mild at the high dose and the relevance is questionable but these are also covered by this NOAEL. There were no effects on fertility. The NOAEL for development toxicity is 2000 ppm (depending on the substance intake during lactation 289.5 mg/kg bw/day) based on reduced litter weights and reduced offspring body weight/body weight gains at 6000 ppm, which can be related to the lower food consumption and food efficiency in the dams.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 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).

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-(p-methoxyphenyl)-2-methylpropionaldehyde

EC Number:

226-749-5

EC Name:

3-(p-methoxyphenyl)-2-methylpropionaldehyde

Cas Number:

5462-06-6

Molecular formula:

C11H14O2

IUPAC Name:

3-(4-methoxyphenyl)-2-methylpropanal

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Age at study initiation: Males: Appr. 11 weeks, Females Appr. 14 weeks
- Weight at study initiation: Males: 283g to 349g, Females: 182g to 233g
- Fasting period before study: no
- Housing: 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. 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.
- Diet: A ground or powdered diet (Rodent PMI 5002 (Certified) Ground Diet (BCM IPS Limited, London, UK)) was provided ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.
- Acclimation period: Eighteen days during which time health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Mar 2017 to 16 May 2017

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): 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/U200 mixer.
- Storage temperature of food: Room temperature, with the exception of the final mix which was prepared for approximately two weeks and split into two weekly aliquots. When in use, the first aliquot was stored at room temperature and the second aliquot, when not in use was kept in labelled, double plastic bags and stored in the freezer (approximately -20 °C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eight days at room temperature and twenty-one days at approximately -20 °C.
- Samples were taken from the dietary admixtures on four occasions and analyzed for uniformity of distribution and concentration of the substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 19% of the nominal concentration.
- The analytical method was based on GC using FID detection

Duration of treatment / exposure:

six weeks (males) or up to eight weeks (females, including a two week pre-pairing phase, pairing, gestation and early lactation)

Frequency of treatment:

Continuous by dietary admixture

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

600 other: ppm (nominal 40 mg/kg bw)

Remarks:

Mean achieved dosage levels: 39.6 mg/kg bw/day (males), 47.1 mg/kg bw/day (females pre-mating), 52.5 mg/kg bw/day (females during gestation) and 96.2 mg/kg bw/day (females during lactation)

Doses / concentrations 2

Dose / conc.:

2 000 other: ppm (nominal 133 mg/kg bw)

Remarks:

Mean achieved dosage levels: 129.7 mg/kg bw/day (males), 161.4 mg/kg bw/day (females pre-mating), 174.1 mg/kg bw/day (females during gestation) and 289.5 mg/kg bw/day (females during lactation)

Doses / concentrations 3

Dose / conc.:

6 000 other: ppm (nominal 400 mg/kg bw)

Remarks:

Mean achieved dosage levels: 389.9 mg/kg bw/day (males), 451.3 mg/kg bw/day (females pre-mating), 553.2 mg/kg bw/day (females during gestation) and 859.8 mg/kg bw/day (females during lactation)

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

Basis for dose level selection: The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work (including a 14 Day range-finding toxicity study in the rat.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioral change once daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS:
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 and Lachrymation.

BODY WEIGHT:
Individual body weights were recorded on Day 1 and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND FOOD EFFICIENCY
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 for the periods covering Days 1-4, 4-7 and 7-14 post partum.
Food efficiency (the ratio of body weight change/dietary intake) and mean achieved dosages were calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation. Mean achieved dosages for females during gestation and lactation have been calculated, however, these values may have been affected by offspring growth during gestation and the possibility of offspring starting to eat the diet during the final week of treatment.

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

HAEMATOLOGY
- Haematological investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 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.
- The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT) and Reticulocyte count (Retic).
- 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).

CLINICAL CHEMISTRY
- Blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 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.
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids and Calcium (Ca++)

THYROID HORMONE ANALYSIS
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into potassium EDTA tubes, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
- Serum and plasma samples were taken from all adult males and females at termination.
- The serum from adult males was analyzed for Thyroxine (T4)

NEUROBEHAVIOURAL EXAMINATION: Functional observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. 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.

NEUROBEHAVIOURAL EXAMINATION: 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.
- 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.

SENSORY REACTIVITY
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex and Finger approach

Sacrifice and pathology:

SACRIFICE
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy were killed around the same time as littering females.

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

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Brain, Spleen, Heart, Kidneys, Thymus, Liver.
- From all remaining animals the following organs where weighted: Pituitary (post-fixation), Prostate and Seminal Vesicles (with Coagulating Gland), Epididymides, Testes, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix and oviducts)

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 otherwise stated: Adrenals, Aorta (thoracic), Muscle (skeletal), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Pancreas, Brain (including cerebrum, cerebellum and pons), Caecum, Rectum, Colon, Salivary glands (submaxillary), Cowpers Glands, Sciatic nerve, Duodenum, Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (in Davidsons fluid), Glans Penis, Spleen, Stomach, Heart, Ileum (including Peyer’s patches), Jejunum, Trachea, Kidneys, Thymus, LABC (levator ani-bulbocavernous) muscle, Urinary bladder, Liver, Lungs (with bronchi, lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative) and Lymph nodes (mandibular and mesenteric).
- For remaining animals the following organs were preserved: Mammary gland, Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides (in Modified Davidsons fluid), Gross lesions, Testes (in Modified Davidsons fluid), Thyroid/Parathyroid, Uterus & Cervix and Vagina.
- The tissues from five selected control and 6000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues as indicated from the remaining control and 6000 ppm animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 6000 ppm males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
- Immunohistochemical staining of the kidneys for α-2u-globulin was also performed (from additional sections from both kidneys) for five selected males from the control and high dose groups.
- Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the spleen from five selected females in the low and intermediate groups.

Statistics:

Please refer to 'Any other information on materials and methods incl. tables'

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent for animals of either sex fed diet containing 600, 2000 or 6000 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males fed diet containing 6000 ppm showed a reduction in body weight gain from Week 3 onwards. Statistical significance was achieved during Weeks 5 (p<0.01) and 6 (p<0.05). Overall body weight gain for these males was 23% lower than controls.
- Females from all dietary groups showed a slight reduction in body weight gain during maturation; however, statistical significance was not achieved and a true dose related response was also not evident, therefore the intergroup differences were considered of no toxicological significance. Body weight gain in all treated females during gestation and lactation was comparable to controls.
- A slight reduction in body weight gain was evident in males fed diet containing 2000 and 600 ppm during Week 3. Statistical significance was not achieved and recovery was evident during Week 4. A statistically significant reduction (p<0.05) in body weight gain was evident for these males during Week 5, however, statistical significance was minimal and a true dose related response was not evident. The intergroup differences evident at 2000 and 600 ppm were therefore considered not to represent a true treatment-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- No adverse effect on food consumption was evident in treated males.
- No adverse effect on food consumption was evident in treated females during maturation or gestation. Females fed diet containing 6000 ppm showed a statistically significant reduction (p<0.001) in food consumption between Days 4 and 7 of lactation. A reduction in food consumption continued for the remainder of lactation (Days 7-14), however, statistical significance was not achieved.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Minor fluctuations in food conversion efficiency were evident in males fed diet containing 6000 ppm during Weeks 5 and 6, however, these were considered to reflect the changes in body weight gain
seen in these males and were considered not to be of toxicological significance.

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

no effects observed

Description (incidence and severity):

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

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the haematology parameters examined. Animals of either sex from all dietary concentrations showed statistically significant increases (p<0.01) in reticulocyte count. All of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
- Males fed diet containing 6000 ppm showed statistically significant reductions in alanine aminotransferase (p<0.05) and cholesterol (p<0.01) and a statistically significant increase in bilirubin (p<0.05). The majority of the individual values were within background control ranges and in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
- Females from all dietary concentrations showed a statistically significant increase in chloride concentration (p<0.01) and a statistically significant reduction in cholesterol (p<0.05-0.01). With the exception of two cholesterol values (one female fed diet containing 2000 ppm and one female fed diet containing 6000 ppm), all of the remaining individual values for cholesterol and chloride concentration were within background control ranges. A true dose related response was also not evident for chloride concentration and no associated histopathological correlates were evident. The intergroup differences were therefore considered not to be of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- There were no treatment-related changes in the behavioral parameters at 600, 2000 or 6000 ppm.
- Sensory reactivity scores across all test item-treated dose groups were similar to controls.
- Functional performance tests: There were no intergroup differences at any dietary concentration considered to be related to treatment with the test item. Males fed diet containing 6000 ppm showed a statistically significant increase in hind limb grip strength (p<0.05-0.01) in two out of the three tests. During motor activity evaluations, males from this dietary group also showed a statistically significant reduction (p<0.05) in the final 20% of activity monitoring time and females showed statistically significant increases (p<0.01) in overall activity and the final 20% of activity monitoring time. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Females fed diet containing 6000 ppm showed a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. Although all of the individual values were within background control ranges, this reduction in weight does correlate with the microscopic changes evident in the spleen of these animals. Therefore, a relationship to treatment cannot be excluded.
- No such effects were detected in males fed diet containing 6000 ppm or animals of either sex fed diet containing 2000 or 600 ppm.
- Females fed diet containing 2000 and 6000 ppm showed a statistically significant increase (p<0.05) in adrenal weights both absolute and relative to terminal body weight. Males treated with 2000 ppm showed a statistically significant reduction (p<0.05) in liver weight both absolute and relative to terminal body weight. All of the individual values were within background control ranges and in the absence of a true dose-related response, the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic findings detected in adult animals. Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included a mottled liver (one control male, one control female and one 2000 ppm female), a pale liver (one 600 ppm female and one 2000 ppm female), mottled kidneys (one control male and one 6000 ppm male) and small flaccid testes and epididymides (one 6000 ppm male).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The following treatment-related microscopic abnormality was detected in the spleen: A reduction in hematopoiesis was evident in females fed diet containing 6000 ppm. All females from the control, 600 and 2000 ppm groups had minimal or mild hematopoiesis as generally seen in lactating females in this type of study. Hematopoiesis was not recorded in any females fed diet containing 6000 ppm. No such effect was evident treated males.
- Special stains of male kidney sections, for the presence of α-2u-globulin were examined and did not indicate significant or consistent differences between control and males fed diet containing 6000 ppm.
- There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Achieved Intake

Group mean achieved dosage of substance in mg/kg bw/day during the study are calculated using nominal concentration for dietary formulations.

- At 6000 ppm, mean achieved dosage for males was 389.9 mg/kg bw/day and for females during the pre-pairing period was 451.3 mg/kg bw/day. Mean achieved intakes were fairly consistent for animals of either sex and generally maintained a 10 fold interval between this high dietary level and the low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 2000 ppm, mean achieved dosage for males was 129.7 mg/kg bw/day and for females during the pre-pairing period was 161.4 mg/kg bw/day. Mean achieved intake was fairly consistent for animals of either sex and generally maintained a 3 fold interval between this intermediate dietary level and low dietary level. As to be expected, mean achieved intake increased during gestation and lactation due to the increased demand on the adult female.

- At 600 ppm, mean achieved dosage for males was 39.6 mg/kg bw/day and for females during the pre-pairing period was 47.1 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period and increased slightly during gestation and lactation due to the increased demand on the adult female.

Applicant's summary and conclusion

Conclusions:

For the substance a parental NOAEL for systemic effects of 2000 ppm was derived based on food efficiency and body weight gain in males and body weight gain, food consumption, reduced spleen weight and reduction in the amount of haematopoiesis in the spleen of high dose females. The lowest derived NOAEL is 129.7 mg/kg bw/day based on the lower food intake in males being the most conservative value of this 2000 ppm dose.

Executive summary:

A study was performed according to OECD TG 422 and GLP principles.The test item was administered by continuous dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dietary concentrations of 600, 2000 and 6000 ppm (nominal 40, 133 and 400 mg/kg bw and equivalent to a mean achieved dosage of 39.6, 129.7 and 389.9 mg/kg bw/day for males and 47.1, 161.4 and 451.3 mg/kg bw/day respectively for females during maturation, 52.2, 174.1 and 553.2 mg/kg bw/day respectively for females during gestation and 96.2, 289.5 and 859.8 mg/kg bw/day respectively for females during lactation). A control group of twelve males and twelve females were treated with basal laboratory diet.

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 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and ano-genital distance and visible nipple count (male offspring only). Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4). Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of termination for all treated females. Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14 post partum, respectively. Any female which did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results Parental toxicity

No mortality or clinical signs were detected. In addition no changes in behavioral parameters, functional performance or sensory activity were observed. Regarding the body weight,males fed diet containing 6000 ppm showed a reduction in body weight gain from Week 3 onwards. Overall body weight gain for these males was reduced when compared to controls. No toxicologically significant effects were evident in males fed diet containing 2000 or 600 ppm. No toxicologically significant effects in body weight gain were evident in treated females during maturation. Body weight gain in all treated females during gestation and lactation was comparable to controls. No adverse effect on food consumption was evident in treated males. Food conversion efficiency was reduced in males fed diet containing 6000 ppm during Weeks 5 and 6 of treatment up to 44% at the end of exposure. No such effect was evident in males fed diet containing 2000 or 600 ppm. No adverse effect on food consumption or food conversion efficiency (maturation only) was evident in treated females during maturation or gestation; however, females fed diet containing 6000 ppm showed a reduction in food consumption between Days 4 and 14 of lactation. No such effect was evident in females fed diet containing 2000 or 600 ppm. Food efficiency (during maturation) was also reduced up to 19%. Daily visual assessment of water consumption did not reveal any significant intergroup differences.

Haematology: There were no toxicologically significant effects detected in the hematological parameters or blood chemistry examined. Upon Necropsy neither the type, incidence or distribution of macroscopic observations in adult animals or offspring indicated any treatment-related effect up to a dietary concentration of 6000 ppm.

Clinical chemistry: No effects were observed

Organ related effects: Absent of increased liver weights or other organs. Females fed diet containing 6000 ppm showed a statistically significant reduction in spleen weight both absolute and relative to terminal body weight. No such effects were detected in males fed diet containing 6000 ppm or animals of either sex fed diet containing 2000 or 600 ppm. A reduction in the amount of hematopoiesis in the spleen of females fed diet containing 6000 ppm was evident when compared to controls. No such effect was evident in any treated male or in females fed diet containing 2000 or 600 ppm.

Hormone analysis: An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Fertility

Regarding the reproductive parameters, there was no effect of treatment with the test item at any dietary concentration on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy. There was no effect of treatment on mating performance. With the exception of five pairings, all animals mated within five days of pairing. There were no treatment-related effects in conception rates for test item-treated animals in relation to controls and no differences in gestation lengths in animals receiving the test item when compared with controls were observed.

Offspring

There was no detrimental effect of treatment with the test item on the mean number of implantations, post-implantation loss, litter size, sex ratio and subsequent offspring survival to Day 13 of age at 600, 2000 and 6000 ppm. Offspring body weight and litter weight on Day 1post partumwas comparable to controls. Offspring body weight, offspring body weight gain and litter weights from females fed diet containing 6000 ppm was reduced on Days 4, 7 and 13 post partum when compared to control litters. No such effects were evident in litters from females fed 2000 or 600 ppm. There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1 post partumat visible nipple count in male offspring on Day 13 post partumat 600, 2000 or 6000 ppm. An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Summary

For systemic toxicity some effects were seen on body weight (gain), food consumption and food efficiency at the high dose. In absence of clear palatability effects these effects are considered adverse at the high dose of 6000 ppm, resulting in a NOAEL of 2000 ppm (129.7 mg/kg bw/day for males and 161.4 mg/kg bw/day for females). The other effects seen in hematology, spleen and in adrenals are mild at the high dose and the relevance is questionable but these are also covered by this NOAEL. There were no effects on fertility, the ‘No Observed Adverse Effect Level’ (NOAEL) for fertitlity is considered to be ≥ 6000 ppm (389.9 mg/kg bw/day, based on the lower intake on males). The NOAEL for development toxicity is 2000 ppm (depending on the substance intake during lactation 289.5 mg/kg bw/day) based on reduced litter weights and reduced offspring body weight/body weight gains at 6000 ppm, which can be related to the lower food consumption and food efficiency in the dams.