3-(p-methoxyphenyl)-2-methylpropionaldehyde

Endpoint summary

Description of key information

A study in accordance with OECD TG 422 reveals a NOAEL for systemic effects of 129.7 mg/kg bw/day for males and 161.4 mg/kg bw/day for females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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 other 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

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

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)

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)

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.

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'

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.

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

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

Key result

Critical 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 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

129.7 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A study is conducted according to OECDTG422 in compliance with GLP. The resulting information is adequate for the endpoint.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 422 Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening

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 postpartum. 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 postpartum, 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 postpartum, 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 adverse 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 1 postpartum 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 postpartum 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 postpartum at visible nipple count in male offspring on Day 13 postpartum at 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 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.

Justification for classification or non-classification

Based on the available information, classification and labelling for repeated exposure is not warranted in accordance with EU CLP (EC No. 1272/2008 and its updates).