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Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test: NOAEL = 887 mg/kg bw/day in female rats; NOAEL = 791 mg/kg bw/day in male rats (OECD 422, GLP, K, rel.1). No adverse effect reported at the highest dose level tested in this study.

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:
11 August 2014 to 13 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 05 February 2014 / signed on 7 October 2014)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of animals at study initiation: Males: ca. 79 days; Females: ca. 72 days
- Weight at study initiation: Males: 334-428 g; Females: 212-275 g
- Housing:
Number of animals per cage:
Pre-pairing, toxicity phase females: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Recovery phase: up to five animals of one sex
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated. A minimum of 15 air changes per hour.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 11 August 2014 to 16 October 2015
Route of administration:
oral: feed
Details on route of administration:
The dietary of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were made weekly, divided into daily aliquots.
- Mixing appropriate amounts with SDS VRF1 certified diet: The required amount of test material with an equal amount of sieved diet was ground to a powder, using a mechanical grinder, in the presence of dry ice to keep the mixture as cool as possible. This powder was then added to an approximately equal amount of sieved diet and stirred. An amount of sieved diet approximately equal to the weight of the mixture was added and the mixture stirred until it appeared homogeneous. This doubling up process with the sieved diet was continued until approximately half of the premix diet had been added. At this stage, the mixture was ground using a mechanical grinder. Coarse diet was then added to make the premix up to the required amount and mixing was continued in a Turbula mixer for 100 revolutions. This ensured the test substance was dispersed in the diet. The premix was then diluted with further quantities of coarse basal diet to prepare the required concentration for each test group.
The test material was not heated and contact with plastics was avoided.
- Storage temperature of food: Frozen until immediately prior to feeding daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: The formulation was proven to be homogenous and stable in the following conditions:
500 ppm - ambient temperature (nominally +21 °C) for 24 hours and refrigerated (nominally +4 °C) and frozen (nominally -20 °C) for 22 days.
20000 ppm - ambient temperature (nominally +21 °C) for four days and refrigerated (nominally +4 °C) and frozen (nominally -20 °C) for 22 days.
Achieved concentrations: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentrations of the test substance (See table 7.5.1.2 and 7.5.1.3 in results and discussion).
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Reproductive phase females: Two weeks before pairing, then throughout pairing, gestation and lactation. For females selected for blood sampling on Day 8 of lactation treated diet was removed on the afternoon of Day 7 of lactation. For females not selected for blood sampling treated diet was removed on the morning of Day 8 of lactation.
Toxicity phase females: At least five weeks.
Recovery animals: At least five weeks (followed by at least 14 days recovery).
Frequency of treatment:
Continuously (ad libitum). Food in the hoppers was changed daily.
Dose / conc.:
1 500 ppm
Dose / conc.:
5 000 ppm
Dose / conc.:
15 000 ppm
No. of animals per sex per dose:
Number of main study animals: 10 animals/sex/dose
Toxicity phase females: 5 animals/dose for control and high dose groups
Recovery phase: 5 animals/sex/dose for control and high dose groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The doses used in this study (0, 1500, 5000 and 15000 ppm) were selected in conjunction with the Sponsor. Dose levels were selected following the completion of a preliminary study (Envigo Study Number HIK0044) conducted at this laboratory. In this study, test substance in the diet was administered to CD rats at the dose levels of 1500, 7500 and 15000 ppm for 7 days. Achieved dose maintained the approximate intervals between dose levels. Mean achieved dose was for males 101, 493 and 834 mg/kg bw/day and for females 127, 521 and 992 mg/kg bw/day for females at dose levels of 1500, 7500 and 15000 ppm respectively. There were no premature deaths, no clinical signs or visual water consumption effects which were attributable to the administration test substance. Body weight gain was slightly low between Days 1-4 of dosing in males receiving 15000 ppm and females receiving 7500 or 15000 ppm. Food consumption was slightly low in males and females receiving 7500 or 15000 ppm following the first day administration only. There was an indication of high liver weight in males receiving 15000 ppm although this would not be considered dose limiting at the degree observed. There was no clear effect of treatment in other organs or the lower dose levels of 7500 or 1500 ppm. There were no changes associated with treatment detected at necropsy. The results of this study indicate
that, the limit dose of 15000 ppm (to achieve a target 1000 mg/kg/day) was selected as the high dose and low and intermediate dose levels of 1500 and 5000 ppm were selected to investigate dose relationships of any potential effects of treatment.
- Rationale for animal assignment: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management.
- Rationale for selecting satellite groups: Subgroups of males and females were used to assess recovery, persistence or delayed occurrence of systemic effects (recovery group; non-paired animals). Another group of non-paired females were treated for at least 5 weeks.
- Post-exposure recovery period in satellite groups: 14 days recovery for the recovery groups only
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, detailed physical examination and arena observations were performed on each appropriate animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations:
Main phase males, recovery phase animals and toxicity phase females: Week -1, on the day that treatment commenced (Week 0) and weekly thereafter.
Main phase females: Week -1, on the day that treatment commenced (Week 0); Weekly before pairing; Days 0, 7, 14 and 20 after mating; Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations:
F0 animals:
- Weekly pretreatment from Day -7.
- Daily, from the commencement of treatment.
- Food consumption was not recorded for main study males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
- Food consumption was recorded continuously for recovery phase males and females and toxicity phase females.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the sublingual vein of animals on Week 5 (The five lowest numbered surviving males per group (Main phase) and females per group (Toxicity phase)) and on Day 8 of lactation (The five lowest numbered surviving females per group (Main phase)).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, Animals were deprived of food and water overnight, but were allowed access to
water for a minimum of 1 hour prior to the commencement of blood sampling. On Day 8 of lactation,
blood samples were collected after overnight withdrawal of food.
- How many animals: 47 (5 animals/group for males (Main phase) and females (Toxicity phase) after Week5 and for main phase females on Day 8; Insufficient samples obtained in two males of the control group and no sample obtained in one female of the mid-dose group)
- Parameters checked : Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count (WBC), Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the sublingual vein of animals on Week 5 (The five lowest numbered surviving males per group (Main phase) and females per group (Toxicity phase)) and on Day 8 of lactation (The five lowest numbered surviving females per group (Main phase)).
- Animals fasted: Yes, Animals were deprived of food and water overnight, but were allowed access to water for a minimum of 1 hour prior to the commencement of blood sampling. On Day 8 of lactation,
blood samples were collected after overnight withdrawal of food.
- How many animals: 47 (5 animals/group for males (Main phase) and females (Toxicity phase) after Week5 and for main phase females on Day 8; Insufficient samples obtained in two males of the control group and no sample obtained in one female of the mid-dose group)
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Blood urea nitrogen (BUN), Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 5 (The lowest numbered surviving males (main phase) and females (Toxicity phase) per group).
- Metabolism cages used for collection of urine: Yes; Animals were placed in an individual metabolism cage, without food or water.
- Animals fasted: Yes
- Parameters checked:
Using manual methods: Clarity and Colour - by visual assessment, Volume - using a measuring cylinder, pH - using a pH meter, Specific gravity - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyser: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal.
- Dose groups that were examined:
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 5 of treatment and on the five lowest numbered lactating main study females in each group at Days 4-6 of lactation.
Motor activity of the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 5 of treatment, and the five lowest numbered lactating main study females in each group at Days 4-6 of lactation, was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson
Technical Services and software developed and maintained by Envigo.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Sacrifice and pathology:
SACRIFICE
- Main phase males and toxicity phase females: After Week 5 investigations completed.
- Main phase females: Day 8 of lactation.
- Recovery phase animals: After at least 14 days without treatment.
All adult animals were killed by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY: Yes (SeeTable 7.5.1/1)
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Main phase females - Each uterine horn and ovary: Number of implantation sites and corpora lutea.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes (SeeTable 7.5.1/1)
- Organ weights: For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.
- Histopathology
Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - In modified Davidson’s fluid; Eyes - In Davidson’s fluid.
Histology
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: The five lowest numbered surviving main phase males, main phase females (with a live litter) and toxicity phase females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All remaining F0 animals.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Light microscopy
Scheduled kill:
Five lowest numbered F0 surviving main phase males and females in Groups 1 and 4 - All specified in Table 7.5.1/1
All remaining main phase animals - Abnormalities only
Toxicity phase females in Groups 1 and 4 - All specified in Table 7.5.1/1
All recovery phase animals - Abnormalities
Other examinations:
None
Statistics:
See section "Any other information on materials and methods incl. tables”.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no dosing signs or changes detected at physical examination or arena observations that were considered to indicate an immediate or delayed reaction to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Group mean body weight gain during the first week of treatment was low for males and body weight loss was recorded for females at 15000 ppm. Body weight gains of main and toxicity phase females receiving 1500 or 5000 ppm were also lower than Control although no dose trend was apparent. After week 0-1 there were no clear trends of impaired body weight performance, however the low overall body weight gain week 0-5 did attain statistical significance for both males and toxicity phase females receiving 15000 ppm. It was noted that toxicity and recovery females had statistically low weight gain for week 1-2. However, as the main study females which were in exactly the same physical state at that time did not present the same response this is considered to be coincidental. No statistical significance was attained in the body weight change in females in the main study phase during the period week 0-2.
- There was no indication of a body weight effect during gestation and body weights on Day 1 of lactation were similar in all groups. Low body weight gain during Days 1-4 of lactation, when compared to Controls was noted in all treated groups but attained statistical significance for animals receiving 15000 ppm only. There was no similar effect during Days 4-7 of lactation.
- During recovery male body weight performance was similar to control. During week 1 of recovery, females previously receiving 15000 ppm, displayed a high weight gain which was followed by body weight loss in the second week of recovery. Overall body weight performance of females during recovery was not affected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Group mean food consumption was marginally low for the first week of treatment for males and females at 15000 ppm. This difference mainly occurred during the first day or two of receiving the treated diet and quickly recovered.
- There was no indication of a food consumption effect during gestation.
- Low food consumption was observed throughout lactation from Day 2 for all groups of treated females, with statistical significance being attained on several occasions (Days 3, 5 and 6) for females receiving 15000 ppm. Differences for females receiving 5000 or 1500 ppm also attained statistical significance on Day 5 only, however, there was generally no clear dose response evident between the 1500 and 5000 ppm dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- At haematology evaluation during week 5 of study there were no statistically significant differences to the Control for males. For females there were no consistent effects on any individual parameters assessed although statistical significance was attained for: short prothrombin time, though only one female was outside the historical Control range (historical control 90 percentile range: 21.37-26.05 x10^9/L) and low haemoglobin concentration (historical control 90 percentile range: 14.08-15.40 g/dL) in toxicity phase females during week 5 of study, these effects were not observed in the main phase females on Day 8 of lactation. On Day 8 of lactation high monocyte count (historical control 90 percentile range: 0.13-0.51 x10^9/L) and high neutrophil count which was within the historical control range (historical control 90 percentile range: 2.29-6.73 x10^9/L) for main phase females receiving 15000 ppm, these differences were not apparent in toxicity phase females. These changes were attributed to natural variation or were considered not adverse at the degree observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no consistent effects on blood chemistry parameters evaluated for males and toxicity phase females during week 5 of study or main phase females on Day 8 of lactation.
- In males only receiving 15000 ppm urea (and blood urea nitrogen) was statistically high but within the historical control range (urea historical control 90 percentile range: 4.72-6.60 mmol/L) and calcium ion concentration (historical control 90 percentile range: 2.37-2.71 mmol/L) was statistically lower than Control. Cholesterol was statistically higher than control but within the historical control range (historical control 90 percentile range: 0.94-1.96 mmol/L) at 15000 or 5000 ppm and a dose response was not apparent. There were no other differences which attained statistical significance at 1500 or 5000 ppm.
- In toxicity phase females during week 5 of study receiving 15000 ppm alkaline phosphatase was low but within the historical control range (historical control 90 percentile range: 43-78 U/L); alanine amino transferase was low (historical control 90 percentile range: 24-49 U/L); urea (and blood urea nitrogen) was low but within the historical Control range (urea historical control 90 percentile range: 5.04-7.76 U/L) and glucose was also low but within the historical control range (historical control 90 percentile range: 6.17-11.48 mmol/L). Statistical significance was attained for these differences however these differences were not apparent in lactating females.
- During lactation statistical significance was attained for high bile acid concentration (historical control 90 percentile range: 10.4-125.0 μmol/L) at 15000 ppm which was within the historical control range and influenced by a single atypically high result in that group (animal No. 119); phosphorus concentration (historical control 90 percentile range: 2.11-3.79 mmol/L) was also higher than Control in all groups of treated females however no dose trend was apparent and all values were within the historical control range; significance was also attained for high potassium (historical control 90 percentile range: 4.3-5.54 mmol/L) in females receiving 15000 ppm values although values were within the historical Control range.
- Above values attaining statistical significance were often within the historical range or close to the Control range, apparent in only one of the populations assessed or were considered not to be adverse at the degree observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no adverse effects on urinary parameters measured during week 5 of study.
- All inter-group differences from controls, including those that attained statistical significance, were minor, generally within the historical control range or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included statistically significant differences in the males only including low creatinine concentrations at 15000 ppm but within the historical control range (historical control 90 percentile range: 62.328-104.388 μmol), low potassium concentration in all dose levels was only slightly outside the historical range at 15000 ppm (historical control 90 percentile range: 1.010-1.923 mmol) a dose dependant trend was apparent, low urinary chloride concentration (historical control 90 percentile range: 0.178-0.563 mmol) at 15000 or 5000 ppm. A similar trend of lower urinary creatinine, potassium than Control was apparent in the females however the difference did not attain statistical significance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- There were no indications of any adverse effects on sensory reactivity or grip strength assessments.
- Motor activity was considered not to be adversely affected by the test material.
- There were no periods for females either non-mated or during lactation during which activity displayed statistically significant differences to the Control. Males did indicate a trend towards slightly lower activity scores, at times attaining statistical significance in animals receiving 15000 or 5000 ppm and on a single occasion 1500 ppm also. The differences lacked an indication of any dose related response, overall activity scores were not statistically different to the Control and most of the statistically significant differences were within the historical Control data range. During the recovery period the assessment was repeated for males and there were no statistical differences to Control.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Terminal body weights were similar in all groups of males and females. When compared to concurrent Controls, there were no differences in male organ weights that attained statistical significance.
- In toxicity phase females adjusted mean liver weight was statistically higher, and the adjusted mean uterus with cervix and oviducts weight was statistically lower, than controls in females receiving 15000 ppm.
- In reproductive phase females these organ did not display a similar effect and only the adjusted mean adrenal weights were statistically lower than Control.
- Following two weeks recovery there were no effects on the weights of any organs assessed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no changes detected at necropsy of the adult animals which indicated a treatment related response to test substance either after 5 weeks of dosing, during lactation or following two weeks recovery.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no treatment related findings.
- Findings of uncertain relationship to treatment: Minimal centrilobular hypertrophy was recorded in the liver of two main study females treated with 15000 ppm and one toxicity phase female treated with 15000 ppm.
- Incidental findings: All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was considered to be no effect on oestrous cycles, pre-coital interval, mating performance, fertility or gestation length or index.
Dose descriptor:
NOAEL
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
Key result
Dose descriptor:
NOAEL
Effect level:
>= 791 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 887 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no

Formulation analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, limit of quantification, linearity of detector response, repeatability, accuracy and precision.

The homogeneity was confirmed for test substance in SDS VRF1 certified diet formulations at nominal concentrations of 500 ppm and 20000 ppm. Stability was confirmed at ambient temperature storage for up to 1 day and frozen (nominally -20 ºC) storage for 22 days.

The mean concentrations of test substance in test formulations analysed for the study were within 12% of nominal concentrations, confirming accurate preparation. Difference from mean values remained within 3%, confirming precise analysis, with the exception of Group 2 in Week 5, which had a difference from mean of ±5.05%.

 

Table 7.5.1/2: Achieved dose (mg/kg/day) for all males and toxicity and recovery phase females

 

Sex

Males

Females

Group

2

3

4

2

3

4

Level

1500

5000

15000

1500

5000

15000

Mean

75.6

258

791

-

-

887

  

Table 7.5.1/3: Achieved dose (mg/kg/day) for main phase females

 

Sex

Females

Group

2

3

4

Level

1500

5000

15000

Mean – before pairing

95.2

320

922

Mean - during gestation

95.5

319

946

Mean – during lactation

181

626

1768

 

Achieved doses generally maintained the intervals between dietary concentrations. The increased achieved intake during lactation reflected the greater physiological demand on the dams.

See the attached document for information on Tables of results 422-repeated dose toxicity

Conclusions:
Under the test conditions, the systemic parental NOAEL was considered to be 15000 ppm in rats (i.e. 791 mg/kg bw/day in male rats and 887 mg/kg bw/day in female rats), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received test substance orally, via the diet, at nominal concentrations of 1500, 5000 or 15000 ppm. Concurrent controls (10 rats/sex) received the basal powder diet without test substance. Main study males (10 animal/dose) were treated for two weeks before pairing and up to necropsy after a minimum of five consecutive weeks. Reproductive main study females (10 animal/dose) were treated for two weeks before pairing, throughout pairing and gestation. Females were allowed to litter, rear their offspring to weaning and were killed on Day 8 of lactation. Toxicity phase females (5 animal/dose) were treated for at least five weeks and were not paired. High dose and control recovery phase male and female rats (5 animal/sex/dose) were treated for a minimum of five consecutive weeks and were retained, without treatment for 14 days (nontreatment period commenced from the first Dam necropsy on Day 7 of lactation), to assess the potential for any treatmentrelated change to recover. Recovery phase animals were not paired. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, visual water consumption, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The accuracy of formulation and the accuracy of analysis were confirmed and procedural recovery values remained within validated limits, confirming the continued accuracy of the method. Mean achieved doses were 75.6, 258 or 791 mg/kg bw/day respectively for males (main phase and recovery phase) receiving 1500, 5000 or 15000 ppm and 887 mg/kg bw/day for females receiving 15000 ppm (toxicity phase and recovery phase). Mean achieved doses before pairing were 95.2, 320 or 922 mg/kg bw/day respectively for main phase females receiving 1500, 5000 or 15000 ppm. During gestation, mean achieved doses were 95.5, 319 or 946 mg/kg bw/day respectively for females receiving 1500, 5000 or 15000 ppm. During lactation, due to increased physiological demand, the mean achieved doses were higher at 181, 626 or 1768 mg/kg bw/day respectively for females receiving 1500, 5000 or 15000 ppm.

The test substance was generally well tolerated at doses up to 15000 ppm, there were no unscheduled deaths. It was considered that there were no treatment related effects on clinical condition, sensory reactivity, grip strength, motor activity, haematology, blood chemistry, urinalysis or macroscopic pathology. 

Group mean body weight gain during the first week of treatment was low for males and body weight loss was recorded for females, receiving 15000 ppm. Group mean food consumption was marginally low for the first week of treatment for males and females receiving 15000 ppm. There was no indication of any latent effect on body weight or food consumption during recovery. There was no indication of any effect on body weight performance or food consumption during gestation. Mean body weight on Day 1 of lactation was similar in all groups. Low body weight gain during Days 1-4 of lactation was noted in all treated group but attained statistical significance for animals receiving 15000 ppm only. Low food consumption was observed throughout lactation from Day 2 for all groups of treated females. These coincided with the expected period of high intake and mean achieved dose at 15000 ppm during lactation was in excess of 1700 mg/kg bw/day 

There were no effects on male organ weights assessed. For toxicity phase females, adjusted mean liver weight was statistically high, and adjusted mean uterus with cervix and oviducts weight was statistically low in females receiving 15000 ppm. In reproductive phase females receiving 15000 ppm, adjusted mean adrenal weights were statistically low. Following two weeks recovery there were no effects on the weights of any organs assessed in males or females.

Histopathology of a full list of tissues revealed minimal centrilobular hepatocyte hypertrophy in the liver of two main study and one toxicity phase females treated with 15000 ppm. The liver weight of the females with this change was generally within the relevant concurrent or historical control organ weight range and there were no consistent effects on blood chemistry or any other parameters to suggest that the liver changes were of toxicological significance. The changes are possibly due to background variation. At this minimal level of severity the liver change is considered non adverse.

 

Under the test conditions, the systemic parental NOAEL was considered to be 15000 ppm in rats (i.e. 791 mg/kg bw/day in male rats and 887 mg/kg bw/day in female rats), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
791 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 1).

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

A key study was identified (Envigo, 2016, Rel. 1). In this Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received test substance orally, via the diet, at nominal concentrations of 1500, 5000 or 15000 ppm.Concurrent controls (10 rats/sex) received the basal powder diet without test substance.Main study males (10 animal/dose) were treated for two weeks before pairing and up to necropsy after a minimum of five consecutive weeks. Reproductive main study females(10 animal/dose)were treated for two weeks before pairing, throughout pairing and gestation. Females were allowed to litter, rear their offspring to weaning and were killed on Day 8 of lactation. Toxicity phase females(5 animal/dose)were treated for at least five weeks andwere not paired. High dose and control recovery phase male and female rats(5 animal/sex/dose)were treated for a minimum of five consecutive weeks and were retained, without treatment for 14 days (nontreatment period commenced from the first Dam necropsy on Day 7 of lactation), to assess the potential for any treatmentrelated change to recover. Recovery phase animals were not paired.During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, visual water consumption, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The accuracy of formulation and the accuracy of analysis were confirmed and procedural recovery values remained within validated limits, confirming the continued accuracy of the method. Mean achieved doses were 75.6, 258 or 791 mg/kg bw/day respectively for males (main phase and recovery phase) receiving 1500, 5000 or 15000 ppm and 887 mg/kg bw/day for females receiving 15000 ppm (toxicity phase and recovery phase). Mean achieved doses before pairing were 95.2, 320 or 922 mg/kg bw/day respectively for main phase females receiving 1500, 5000 or 15000 ppm. During gestation, mean achieved doses were 95.5, 319 or 946 mg/kg bw/day respectively for females receiving 1500, 5000 or 15000 ppm. During lactation, due to increased physiological demand, the mean achieved doses were higher at 181, 626 or 1768 mg/kg bw/day respectively for females receiving 1500, 5000 or 15000 ppm.

The test substancewas generally well tolerated at doses up to 15000 ppm, there were no unscheduled deaths. It was considered that there were no treatment related effects on clinical condition, sensory reactivity, grip strength, motor activity, haematology, blood chemistry, urinalysis or macroscopic pathology. 

Group mean body weight gain during the first week of treatment was low for males and body weight loss was recorded for females, receiving 15000 ppm. Group mean food consumption was marginally low for the first week of treatment for males and females receiving 15000 ppm. There was no indication of any latent effect on body weight or food consumption during recovery.There was no indication of any effect on body weight performance or food consumption during gestation. Mean body weight on Day 1 of lactation was similar in all groups. Low body weight gain during Days 1-4 of lactation was noted in all treated group but attained statistical significance for animals receiving 15000 ppm only. Low food consumption was observed throughout lactation from Day 2 for all groups of treated females. These coincided with the expected period of high intake and mean achieved dose at 15000 ppm during lactation was in excess of 1700 mg/kg bw/day 

There were no effects on male organ weights assessed. For toxicity phase females, adjusted mean liver weight was statistically high, and adjusted mean uterus with cervix and oviducts weight was statistically low in females receiving 15000 ppm. In reproductive phase females receiving 15000 ppm, adjusted mean adrenal weights were statistically low. Following two weeks recovery there were no effects on the weights of any organs assessed in males or females.

Histopathology of a full list of tissues revealed minimal centrilobular hepatocyte hypertrophy in the liver of two main study and one toxicity phase females treated with 15000 ppm. The liver weight of the females with this change was generally within the relevant concurrent or historical control organ weight range and there were no consistent effects on blood chemistry or any other parameters to suggest that the liver changes were of toxicological significance. The changes are possibly due to background variation. At this minimal level of severity the liver change is considered non adverse.

 

Under the test conditions, the systemic parental NOAEL was considered to be 15000 ppm in rats (i.e. 791 mg/kg bw/day in male rats and 887 mg/kg bw/day in female rats), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding specific target organ toxicity after oral dose-repeated exposure.