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Description of key information

Oral (dietary): NOAEL (rat): Male: 2500 ppm (equivalent to ≥ 146 mg/kg bw/day), and/or Female: 12000 ppm (equivalent to ≥ 806 mg/kg bw/day male/female), OECD TG 422, 2019

The observed hyaline droplet accumulation due to alpha-2u-globulin, is both sex and species specific and is not generally considered to be significant in man.

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:
26-10-2018 to 23-04-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: February 2019 ; signature: August 2019
Limit test:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-diet formulation: Refrigerated (2 to 8°C), in the dark under nitrogen
(ii) Formulated-diet: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test. In the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 4 days when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable. The test item was directly prepared into formulated diet. By the following:
the test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The required amount of test item was weighed into a suitable container. An amount of diet that approximately equalled the weight of test item was added and the mixture stirred together. A further amount of diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder, after which it was made up to the final weight of the premix with diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable.
- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not applicable. Applied in formulated diet.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.
Species:
rat
Strain:
other: RccHan ; WIST
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 422 relevant guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males 86 to 92 days old ; females 100 to 106 days old.
- Weight at study initiation: males 312 to 365 g ; females 191 to 245 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: eight days before commencement of treatment; females: 22 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 Certified powdered diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 55 ± 15 (or 40 to 70)
- Air changes (per hr): Fresh filtered air was passed and not recirculated (air changes per hr, not reported)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2019-12-12 To: 2019-02-15
Route of administration:
oral: feed
Details on route of administration:
The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The required amount of test item was weighed into a suitable container. An amount of diet that approximately equalled the weight of test item was added and the mixture stirred together. A further amount of diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder, after which it was made up to the final weight of the premix with diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified).
- Storage temperature of food: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test. In the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 4 days when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL and 60 μg/mL dissolved into acetonitrile, within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 0.0871 μg/mL and LOQ = 0.290 μg/mL; linearity = > 0.999 between 10 μg/mL and 60 μg/mL. Repeatability (n=6) of < 1%. Accuracy and precision was confirmed and mean procedural recovery was 104.8% (n=5 ; CV = 0.73%) at 500 ppm and 99.7% (CV = 0.31; n=5) at 12000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet at nominal concentrations of 500 ppm and 12000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 8 days and ambient/frozen temperature (-10 to -30°C) for up to 28 days (1 day ambient then 21 days frozen) following fresh preparation. In addition, stability was also confirmed for 4 days ambient plus 4 days frozen storage, 4 days ambient plus 15 days frozen storage and for 22 days frozen storage. At each time-point, the mean analyzed concentration for the two samples remained at or within 10% of the initial time zero value and the difference from mean was less than 2%. Although the relative mean error from nominal was within acceptance limits for the additional time zero value and the relative mean error from time zero values for Day 6 and Day 8 ambient storage were within acceptance limits, the low level formulation was -14.0% from time zero for the time zero timepoint and -19.0% and -19.4% from nominal concentration for the Day 6 and Day 8 timepoints respectively. Therefore, the decision was taken within the study, that the Day 6 and Day 8 ambient stability results would not be accepted because the concentrations were too far from nominal. It was agreed that 4 days ambient stability and 22 days frozen stability would be accepted for this study.
- Mean concentrations of diet-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/diet formulation. Specifically, samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item. The mean concentrations of test item were within 8% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 4%, confirming precise analysis.
Duration of treatment / exposure:
Toxicity phase and recovery groups: Two weeks pre-paring after minimum of 5 weeks treatment: as 5 weeks exposure and 2 week recovery period.
Reproductive phase groups: Minimum of two weeks of treatment and two weeks pairing and gestation until day 13 lactation.
Frequency of treatment:
Continuous via diet
Dose / conc.:
0 ppm
Remarks:
Control – Group 1; Basal diet
Dose / conc.:
2 500 ppm
Remarks:
Low – Group 2
Dose / conc.:
6 000 ppm
Remarks:
Intermediate – Group 3
Dose / conc.:
12 000 ppm
Remarks:
High – Group 4
Dose / conc.:
146 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 2500 ppm group
Dose / conc.:
342 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 6000 ppm group
Dose / conc.:
667 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 12000 ppm group
Dose / conc.:
177 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 2500 ppm group
Dose / conc.:
420 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 6000 ppm group
Dose / conc.:
806 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 12000 ppm group
No. of animals per sex per dose:
Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
2500 ppm (toxicity test) = 10
6000 ppm (toxicity test) = 10
12000 ppm (toxicity test) = 5
12000 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
2500 ppm (reproduction test) = 10
2500 ppm (toxicity test) = 5
6000 ppm (reproduction test) = 10
6000 ppm (toxicity test) = 5
12000 ppm (reproduction test) = 10
12000 ppm (toxicity test) = 5
12000 ppm (recovery period) = 5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 2500, 6000 and 12000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report). Test item was exposed at dietary concentrations of 0 (control), 5000, 10000 and 20000 ppm respectively. It was determined that 20000 ppm would be unsuitable for a subsequent toxicity study.
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ±20% of the mean for the appropriate sex and/or due to poor condition or ill health.
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during acclimatisation at least once daily ; during treatment at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: inspected visually at least twice daily for evidence of ill-health or reaction to treatment. During Littering Phase: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole. Arena observations were conducted during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, on each individual.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 Toxicity and Recovery phase males and females: Weekly during acclimatisation, before feeding of the treated diets on the Day that treatment commenced (Day 1) and twice weekly thereafter (including recovery and day of termination). F0 Reproductive phase females: Weekly during acclimatization. Before the feeding of treated diets on the Day that treatment commenced and weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Mean daily consumption per animal (g/animal/day) was calculated for each phase
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Achieved dose was calculated using the mid-period body weight.
- Other: Food consumption was recorded for F0: Daily (including recovery phase). Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 21) but recommenced for males on Day 21. Reproductive females after mating food consumption was recorded daily until Day 13 of lactation.

FOOD EFFICIENCY: Yes.
- 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 AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study)
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: Pre-treatment (including spares). At Week 5, all surviving Toxicity and Recovery phase animals from Groups 1 and 4 and the five lowest numbered surviving males and females in the Toxicity phase from Groups 2 and 3.
- Dose groups that were examined: All groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Peripheral blood samples: All toxicity groups at termination and recovery groups week 2. For reproductive phase: Day 14 of lactating (females) and test termination (males and females).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No.
- How many animals: five lowest numbered surviving toxicity males per group. All Toxicity phase females. All recovery males/females. All reproductive phase females.
- Parameters checked:
Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt). Additionally: Prothrombin time (PT) was assessed using IL PT Fibrinogen reagent and Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples: All toxicity groups at termination and recovery groups week 2. For reproductive phase: Day 14 of lactating (females) and test termination (males and females).
- Animals fasted: No.
- How many animals: five lowest numbered surviving toxicity males per group. All Toxicity phase females. All recovery males/females. All reproductive phase females.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein. Albumin/globulin ratio (A/G Ratio) (by calculation).

URINALYSIS: Yes.
- Time schedule for collection of urine: five lowest numbered surviving toxicity phase males and females (Week 5) and all recovery phase (Recovery Week 2)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight).
- Parameters checked: Clarity and Color (App) - by visual assessment, Volume (Vol) - using a measuring cylinder, pH - using a pH meter, Specific gravity (SG), Ketones (Keto), Bile pigments (Bili), Urobilinogen (Urob), Blood pigments (UBld), Protein total output (T-Prot)*, Protein concentration (Prot), Creatinine total output (T-Creat)*, Creatinine concentration (U-Creat), Glucose total output (T-Gluc)*, Glucose concentration (U-Gluc), Sodium (T-Na), Potassium (T-K), Chloride (T-Cl) [where * = derived value) ; Other abnormal components (A)#, Epithelial cells (Epi)#, Leucocytes (WBC)#, Erythrocytes (RBC)#, Casts# (where #= not performed in recovery phase).

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Arena observations were conducted during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, on each individual. Detailed examination on sensory activity / grip strength / motor activity was conducted as below in week 5 and in lactation.
- Dose groups that were examined: five lowest numbered surviving Toxicity phase males in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 5 of treatment, and the first five lactating Reproductive phase females in each group at Days 7-9 of lactation
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

ESTROUS CYCLE: Yes
- Dry smears – Reproductive females only: taken from beginning of treatment until pairing
- Wet smears – All females (including spares): taken for 14 days before treatment; females that failed to exhibit 4-5 day cycles were not allocated to the study ; Reproductive females only: after pairing until mating ; All females: four days before scheduled termination

THYROID HORMONE ANALYSIS: Yes
- Time schedule:
(i) at day 4 of age, F1 offspring, two females per litter (where possible) - no females were selected when total litter size dropped below ten/litter or if the resultant number of live females were to be less than 3 for subsequent day 13 procedures. One F1 female for T3/T4 (serum) and One F1 female for TSH (plasma).
(ii) At day 13 of age, F1 offspring, two males and two females per litter (where possible) – one male and one female for T3/T4 (serum) where possible and one male and one female for TSH (plasma) where possible
(iii) at the end of treatment: males from toxicity phase and recovery phase
(iv) at termination: Recovery phase F0 males and all recovery phase F0 females surviving to scheduled termination
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides (left and right), Spleen, Heart, Testes (left and right), Kidneys, Thymus, Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with oviducts)
For reproductive phase females: Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.
For offspring: Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. On day 4: F1 externally abnormal offspring examined and abnormal tissues retained. On day 13: All F1 animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.

HISTOPATHOLOGY: Yes, in five lowest numbered surviving toxicity study males, all toxicity phase females, all recovery phase animals and all adult decedents
- Organs and tissues preserved in neutral buffered 10% formalin or Davidson’s fluid (testes, initially and eyes) as applicable: Abnormalities, Adrenals, Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from 2 lobes), Lungs (section from two major lobes including bronchi), Lymph nodes - left axillary and mesenteric, Ovaries, Peyer’s Patch, Prostate, Sciatic nerve, Seminal vesicles with coagulating glands, Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical level), Spleen, Sternum (with marrow), Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus with cervix (weighed with oviducts), Vagina
Microscopic analysis was conducted thereof. Any macroscopically observed abnormalities or lesions were also processed. Including in reproductive phase.
- Other: Further information in attached tables.
- Immunohistochemistry - Kidneys : All Toxicity phase male animals from Group 1 and 4 were assessed for immunostaining for alpha-2u globulin.
Statistics:
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Litter size, survival indices and sex ratio
Ano-genital distance
Organ weights, both absolute and adjusted for terminal body weight

Comparisons were performed:
Group 1 vs 2, 3 and 4 during treatment, gestation and lactation phases
Group 1 vs 4 during recovery

Typical statistical analysis included:
parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937)
For all other comparisons the F1 approximate test was applied. Which included:
parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972)
If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead

Additional tests, included Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953), Wilcoxon rank sum tests (Wilcoxon 1945) and/or Shirley's test (Shirley 1977) and Steel's test (Steel 1959), as appropriate.

For litter size and survival indices, Fisher’s exact tests (Fisher 1973).

Sex ratio were analyzed equivalent to Lipsitz (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.
Mortality:
no mortality observed
Description (incidence):
There were no treatment related mortalities.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Group mean body weight gains throughout the treatment period (Days 1 to 43) for the Toxicity and Recovery phase males that received test item at 2500, 6000 or 12000 ppm were low compared to Control with a dose response apparent (87%, 76% and 61% of Control respectively) with statistical significance at 6000 and 12000 ppm. During the Recovery phase, group mean body weight gain for both males and females that had received 12000 ppm during the treatment phase, was higher than Control although the group mean body weight on Day R15 for the high dose males and females remained lower than Control.

Females:
Group mean body weight gains throughout the treatment period (Day 1 to 43) for the Toxicity and Recovery phase females were moderately low for females that received 12000 ppm (71% of Control) and slightly low for females that received 2500 ppm (86% of Control), however, overall group mean body weight gain for females that received 6000 ppm was comparable to Control. During the Recovery phase, group mean body weight gain for both males and females that had received 12000 ppm during the treatment phase, was higher than Control although the group mean body weight on Day R15 for the high dose males and females remained lower than Control.

Females in gestation:
Group mean body weight gain for Reproductive phase females during gestation (Day 0 to 20) was slightly lower than Control for females that received 6000 or 12000 ppm. During gestation for females that received 2500 ppm was comparable to Control. For Reproductive phase females during lactation, group mean body weight gain was slightly higher than Control for all treated groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Achieved doses generally maintained the intervals between dietary concentrations and increased during lactations due to increased physiological demand.

Males:
An initial reduction in food intake was observed in all treated groups of males and females following the commencement of treatment (Days 1 to 5), with a marked reduction at 12000 ppm and a dose response apparent in both sexes. After the initial reduction, the food intake of all groups of treated Toxicity and Recovery phase males remained lower than Control throughout the treatment period. Group mean food intake during the Recovery period for both males and females that had received 12000 ppm during the treatment period was generally comparable to Control.

Females:
An initial reduction in food intake was observed in all treated groups of males and females following the commencement of treatment (Days 1 to 5), with a marked reduction at 12000 ppm and a dose response apparent in both sexes. The food intake of all treated groups of Toxicity and Recovery phase females throughout the treatment period from Day 5, and all treated groups of Reproductive phase females prior to pairing was generally similar to or slightly lower than Control. Group mean food intake during the Recovery period for both males and females that had received 12000 ppm during the treatment period was generally comparable to Control.

Females in gestation:
Group mean food intake of all treated groups of Reproductive phase females during gestation was marginally lower than Control. A similar trend was observed for Reproductive phase females during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water intake.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology investigations of toxicity phase animals after 6 weeks of dietary administration revealed no treatment related changes in either sex.

There was a statistically significant reduction in prothrombin time for all groups of treated males compared to Control, however as this change was not dose related, was confined to one sex and not associated with any changes to other clotting factors it is considered that this difference represents normal biological variation. There were statistically significant reductions in group mean white blood cell numbers in females that received 6000 or 12000 ppm, however, these were considered to be largely as a result of one atypically high Control value.

Hematology investigations of Reproductive phase females on Day 13 of lactation revealed no treatment related changes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Blood chemistry investigations of Toxicity phase animals after 6 weeks of treatment revealed a dose related increase in urea for males and females and total protein levels for males with statistical significance attained for both parameters in males that received 12000 ppm. Following a 2-week recovery period, urea levels for animals that received 12000 ppm were comparable to Control in both sexes. Total protein levels in males that received 12000 ppm were comparable to Control following a 2-week recovery period. There was a slight dosage-related increase in cholesterol levels in both sexes with statistical significance attained in males that received 6000 or 12000 ppm. Following a 2-week recovery period cholesterol levels in animals that received 12000 ppm were comparable to Control.

Females:
Blood chemistry investigations of Toxicity phase animals after 6 weeks of treatment revealed a dose related increase in urea for males and females. Following a 2-week recovery period, urea levels for animals that received 12000 ppm were comparable to Control in both sexes. Total protein levels in females that received 12000 ppm were statistically significantly higher than Control. There was a slight dosage-related increase in cholesterol levels in both sexes without statistical significance in females. Following a 2-week recovery period cholesterol levels in animals that received 12000 ppm were comparable to Control. A slight dosage-related reduction in phosphorous levels was observed in females with statistical significance attained at 12000 ppm. Following a 2-week recovery period phosphorous levels remained lower than Control for females that received 12000 ppm, however statistical significance was not attained.

Females in gestation:
Blood chemistry investigations of Reproductive phase females on Day 13 of lactation showed a statistically significant decrease in creatinine levels in females that received 12000 ppm, however there was no dose response apparent. There was also a statistically significant increase in triglycerides in females that received 12000 ppm, however there was no dose response apparent.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males:
Urinalysis investigations performed during Week 5 of treatment revealed a dosage-related increase in the total protein output in males, with statistical significance attained at 6000 and 12000 ppm. The protein concentration was also statistically significantly higher than Control in males that received 12000 ppm, however, no dose response was apparent. There was a dosage-related increase in the total glucose output in males, with statistical significance attained at 6000 and 12000 ppm. Glucose concentration was statistically higher than Control in males that received 12000 ppm, however, no dose response was apparent. There was a slight but dosage-related decrease in total creatinine output and creatinine concentration in males, compared to Control, with statistical
significance attained for creatinine concentration at 6000 and 12000 ppm. Urinalysis was investigated again during Week 2 of recovery and this revealed that in both males and females that had previously received 12000 ppm, total protein output, protein concentration, total glucose output, glucose concentration, total creatinine output and creatinine concentration were comparable to Control.

Females:
The total glucose output of all female treated groups was higher than Control, however, no dose response was apparent. Urinalysis was investigated again during Week 2 of recovery and this revealed that in both males and females that had previously received 12000 ppm, total protein output, protein concentration, total glucose output, glucose concentration, total creatinine output and creatinine concentration were comparable to Control.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity and hindlimb grip strength, motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

Forelimb grip strength values during Week 5 of treatment for both Toxicity phase males and females that received test item at 12000 ppm were slightly low compared to Control, with statistical significance attained in the males. The forelimb grip strength values for both males and females fell below the HCD minimum. This can be explained by two males and two females that were reluctant to grip the forelimb bar and subsequently had lower mean forelimb grip strength values which reduced the overall group mean.

Grip strength assessment was repeated on Recovery phase animals during Week 2 of recovery. This assessment revealed that the group mean forelimb grip strength values for males and females that received 12000 ppm were once again low compared to Control and statistical significance was attained for the male value. Three of the four animals that were reluctant to grip the forelimb bar during Week 5 also showed the same reluctance during Week 2 of recovery which again reduced the overall group mean. Therefore, the differences in forelimb grip strength observed during Week 5 of treatment and during Week 2 of recovery are of uncertain relationship to treatment. Grip strength of lactating females was unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
Group mean body weight adjusted liver weights increased in both sexes with statistical significance attained in all treated groups of males. The greatest magnitude of change from Control was observed in males that received 6000 or 12000 ppm. After 2 weeks of recovery, mean adjusted liver weights were comparable to Control in animals that had previously received 12000 ppm. A dose-related increase in adjusted kidney weights was observed in the Toxicity phase males with statistical significance attained at 12000 ppm. After 2 weeks of recovery, group mean adjusted kidney weights for males that had previously received 12000 ppm were comparable to Control.

Group mean body weight adjusted testes weights in all treated groups of males were higher than Control and group mean adjusted combined seminal vesicle with coagulating gland weight in males that received 12000 ppm were slightly lower than Control, however statistical significance was not attained and no dose response was apparent. The group mean adjusted testes and combined seminal vesicle and coagulating gland weights in Recovery phase males that previously received 12000 ppm were comparable to Control after 2 weeks of recovery.

Females:
The group mean adjusted kidney weights of all treated groups of toxicity phase females were slightly higher than Control, however, not to the same extent as the males and with no dose response apparent. After 2 weeks of recovery, group mean adjusted kidney weights for males that had previously received 12000 ppm were comparable to Control, however the mean adjusted female kidney weights remained statistically significantly higher than Control.

For Toxicity phase females that received 12000 ppm, the group mean body weight adjusted combined weight of the uterus, cervix and oviducts appeared low compared to Control, however, there was a strong correlation between the combined weight of the uterus, cervix and oviducts and stage of estrous at termination and therefor there was considered to be no relationship to treatment. In addition, there was a dosage-related decrease in the combined weight of the uterus, cervix and oviducts in the Reproductive phase females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no significant macroscopic/pathological findings observed that were considered related to treatment or were observed in the treatment and recovery phase groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males:
Microscopic examination performed after 6 weeks of treatment revealed test item-related findings (hyaline droplet accumulation) in the kidneys of all treated Toxicity phase males. Nephropathy and an increase in tubular basophilia, compared to Control, were observed in Toxicity phase males at 6000 or 12000 ppm. Microscopic examination performed after 2 weeks of recovery revealed test item-related findings (nephropathy, hyaline droplet accumulation and an increase in tubular basophilia) in the kidneys of several males that previously received 12000 ppm.

Females:
There were no test item-related findings at microscopic examination for Toxicity phase females.

Test item-related histopathological changes (nephropathy) in the kidneys of Toxicity phase males that received 6000 or 12000 ppm were considered to be adverse, with associated increases in body weight adjusted male kidney weights, a dosage-related increase in group mean urea concentration, high total protein concentration in males that received 12000 ppm, a dosage-related increase in total protein output and total glucose output in the urine, an increase in protein concentration and glucose concentration in the urine of males that received 12000 ppm, a dosage-related decrease in the total creatinine output and a non-dose-dependent decrease in creatinine concentration in the urine. Hyaline droplets are a common finding in the kidneys of untreated male rats. The increase in hyaline droplets observed in the kidneys of treated males is consistent with a test item associated increase in accumulation of alpha-2u-globulin. Hyaline droplet accumulation due to alpha-2u-globulin is both sex and species specific and is not generally considered to be significant in man.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected. There were no microscopic neoplastic findings that were considered to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
1. Thyroid Hormone Assessment:
F0 Males: analyses of samples for thyroxine (T4) did not reveal any differences that could be attributed to treatment, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary.
F1 Female offspring: analyses of samples for thyroxine (T4) did not reveal any differences that could be attributed to treatment, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 146 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 806 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males is considered to be 2500 ppm which was equivalent to actual dose received: 146 mg/kg body weight per day. The no-observed-adverse-effect level (NOAEL) for females is considered to be 12000 ppm which was equivalent to actual dose received: 806 mg/kg body weight per day. The test item related histopathological changes (nephropathy) in the kidneys of males, were considered within the study to be adverse. However, Hyaline droplets are a common finding in the kidneys of untreated male rats. The increase in hyaline droplets observed in the kidneys of treated males is consistent with a test item associated increase in accumulation of alpha-2u-globulin. Hyaline droplet accumulation due to alpha-2u-globulin is both sex and species specific and is not generally considered to be significant in man.
Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least five weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received dietary test item at doses of 0 (Control), 2500, 6000 or 12000 ppm test item. Ten males were treated in the 2500 and 6000 ppm doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 12000 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 2500, 6000 or 12000 ppm test item. Toxicity phase males were treated for two weeks before pairing up to necropsy after at least five weeks. Toxicity phase females were treated for at least five weeks. Recovery phase males were treated for two weeks before pairing up to necropsy after at least five weeks followed by a 2-week recovery period. Recovery phase females were treated for at least five weeks followed by a 2-week recovery period. Reproductive phase females were treated for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 2500, 6000 or 12000 ppm for the toxicity and recovery phase were 146, 342 and 667 mg/kg bw/day in males and 177, 420 and 806 mg/kg bw/day in females, respectively.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no effect on clinical condition, sensory reactivity and (hindlimb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. There was no effect on water intake, ophthalmoscopic findings and no toxicologically significant changes in haematological parameters. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult Toxicity phase males or in the Day 13 male and female offspring. Forelimb grip strength values during Week 5 of treatment for both Toxicity phase males and females at 12000 ppm were slightly low compared to Control, with statistical significance in males. Forelimb grip strength values for both males and females fell below the HCD minimum. This can be explained by two males and two females in the high dose group that were reluctant to grip the forelimb bar and had lower mean forelimb grip strength values which reduced the group mean. Grip strength assessment on Recovery phase males/females during Week 2 of recovery indicated mean forelimb grip strength values for males and females at 12000 ppm were again low. The differences in forelimb grip strength observed during Week 5 of treatment and during Week 2 of recovery are of uncertain relationship to treatment. Grip strength of lactating females was unaffected by treatment. An initial body weight loss was observed in all treatment group males and females following commencement, with a dose response. This was followed by weight gain in all groups by Day 3. Group mean body weight gains throughout treatment for Toxicity and Recovery phase males at 2500, 6000 or 12000 ppm were low compared to Control with dose response. After the initial body weight loss, group mean body weight gains throughout the treatment period for Toxicity and Recovery phase females were moderately low at 12000 ppm and slightly low at 2500 ppm. Overall group mean body weight gain for females at 6000 ppm was comparable to Control. Group mean body weight gain for Reproductive phase females during gestation was slightly lower than Control at 6000 or 12000 ppm. For Reproductive phase females during lactation, group mean body weight gain was slightly higher than Control for all treated groups. During the Recovery phase, group mean body weight gain for males and females at 12000 ppm during treatment, was higher than Control although group mean body weight on Day R15 for the high dose males and females remained lower than Control. Blood chemistry investigations of Toxicity phase groups revealed dose related increase in urea for males and females and total protein levels for males with statistical significance in males at 12000 ppm. Following recovery, urea levels were comparable to Control in both sexes. Total protein levels in males at 12000 ppm were comparable to Control following a recovery, however total protein levels in females at 12000 ppm were higher than Control with statistical significance . There was a slight dose-related increase in cholesterol levels in both sexes with statistical significance in males at 6000 or 12000 ppm. Following 2-week recovery cholesterol levels at 12000 ppm were comparable to Control. A slight dose-related reduction in phosphorous levels was seen in females at 12000 ppm. Following recovery phosphorous levels remained lower than Control at 12000 ppm for females, without statistical significance. One female at 12000 ppm had an atypically low level. Blood chemistry investigations of Reproductive phase females on Day 13 of lactation showed a statistically significant decreases in creatinine levels in at 12000 ppm, without dose response. There was also a statistically significant increase in triglycerides in females at 12000 ppm, without dose response. Urinalysis investigations performed during Week 5 of treatment revealed dose-related increase in the total protein output in males, with statistical significance at 6000 and 12000 ppm. The protein concentration was also statistically significantly higher than Control in males at 12000 ppm, without dose response. There was a dose-related increase in the total glucose output in males, with statistical significance at 6000 and 12000 ppm. Total glucose output of all female treated groups was higher than Control, without dose response. Glucose concentration was statistically higher than Control in males at 12000 ppm, without dose response. There was a dose-related decrease in total creatinine output and creatinine concentration in males, with statistical significance for creatinine concentration at 6000 and 12000 ppm. Urinalysis was investigated during Week 2 of recovery in both males and females at 12000 ppm, total protein output, protein concentration, total glucose output, glucose concentration, total creatinine output and creatinine concentration were comparable to Control. Following 6 weeks of treatment, there dose-related decreases in terminal body weights for males with statistical significance at 12000 ppm. Terminal body weights for females at 12000 ppm, were low compared to Control with statistical significance. Group mean body weight adjusted liver weights increased in both sexes with statistical significance in all treatment group males and greatest magnitude at 6000 or 12000 ppm. After 2 weeks of recovery, mean adjusted liver weights were comparable to Control at 12000 ppm. A dose-related increase in body weight adjusted kidney weights was observed in the Toxicity phase males with statistical significance at 12000 ppm. Group mean adjusted kidney weights of all treated groups of Toxicity phase females were slightly higher than Control, not to the same extent and with no dose response. After 2 weeks of recovery, group mean adjusted kidney weights for males at 12000 ppm were comparable to Control. Mean adjusted female kidney weights remained higher than Control. Microscopic examination revealed test item-related findings (hyaline droplet accumulation) in the kidneys of all treated Toxicity phase males. Nephropathy and an increase in tubular basophilia, compared to Control, were observed in males at 6000 or 12000 ppm. Microscopic examination performed after 2 weeks of recovery revealed test item-related findings (nephropathy, hyaline droplet accumulation and an increase in tubular basophilia) in the kidneys of several males at 12000 ppm. There were no test item-related findings at microscopic examination for Toxicity phase females.

 

Conclusion:

Dietary administration of test item to Han Wistar rats for 6 weeks at concentrations up to and including 12000 ppm was generally well tolerated by the Reproductive phase females and their offspring. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was 12000 ppm (mean achieved doses; 838 mg/kg bw/day during gestation and 1763 mg/kg bw/day during lactation). Test item-related histopathological changes (nephropathy) in the kidneys of Toxicity phase males at 6000 or 12000 ppm were considered to be adverse, with associated increases in body weight adjusted male kidney weights, a dose-related increase in group mean urea concentration, high total protein concentration in males at 12000 ppm, a dose-related increase in total protein output and total glucose output in the urine, an increase in protein concentration and glucose concentration in the urine of males at 12000 ppm, a dose-related decrease in the total creatinine output and a non-dose-dependent decrease in creatinine concentration in the urine. Hyaline droplets are a common finding in the kidneys of untreated male rats. The increase in hyaline droplets observed in the kidneys of treated males is consistent with a test item associated increase in accumulation of alpha-2u-globulin. Hyaline droplet accumulation due to alpha-2u-globulin is both sex and species specific and is not generally considered to be significant in man. Based on these findings, the NOAEL for general systemic toxicity via the dietary route was concluded to be 2500 ppm (mean achieved dose: 146 mg/kg bw/day) in male rats and 12000 ppm (mean achieved dose: 806 mg/kg bw/day) in female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
146 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

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

Mode of Action Analysis / Human Relevance Framework

Not applicable.

Additional information

Repeated dose - Oral:

Key study : OECD TG 422, 2019 : The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least five weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received dietary test item at doses of 0 (Control), 2500, 6000 or 12000 ppm test item. Ten males were treated in the 2500 and 6000 ppm doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 12000 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 2500, 6000 or 12000 ppm test item. Toxicity phase males were treated for two weeks before pairing up to necropsy after at least five weeks. Toxicity phase females were treated for at least five weeks. Recovery phase males were treated for two weeks before pairing up to necropsy after at least five weeks followed by a 2-week recovery period. Recovery phase females were treated for at least five weeks followed by a 2-week recovery period. Reproductive phase females were treated for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 2500, 6000 or 12000 ppm for the toxicity and recovery phase were 146, 342 and 667 mg/kg bw/day in males and 177, 420 and 806 mg/kg bw/day in females, respectively.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no effect on clinical condition, sensory reactivity and (hindlimb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. There was no effect on water intake, ophthalmoscopic findings and no toxicologically significant changes in haematological parameters. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult Toxicity phase males or in the Day 13 male and female offspring. Forelimb grip strength values during Week 5 of treatment for both Toxicity phase males and females at 12000 ppm were slightly low compared to Control, with statistical significance in males. Forelimb grip strength values for both males and females fell below the HCD minimum. This can be explained by two males and two females in the high dose group that were reluctant to grip the forelimb bar and had lower mean forelimb grip strength values which reduced the group mean. Grip strength assessment on Recovery phase males/females during Week 2 of recovery indicated mean forelimb grip strength values for males and females at 12000 ppm were again low. The differences in forelimb grip strength observed during Week 5 of treatment and during Week 2 of recovery are of uncertain relationship to treatment. Grip strength of lactating females was unaffected by treatment. An initial body weight loss was observed in all treatment group males and females following commencement, with a dose response. This was followed by weight gain in all groups by Day 3. Group mean body weight gains throughout treatment for Toxicity and Recovery phase males at 2500, 6000 or 12000 ppm were low compared to Control with dose response. After the initial body weight loss, group mean body weight gains throughout the treatment period for Toxicity and Recovery phase females were moderately low at 12000 ppm and slightly low at 2500 ppm. Overall group mean body weight gain for females at 6000 ppm was comparable to Control. Group mean body weight gain for Reproductive phase females during gestation was slightly lower than Control at 6000 or 12000 ppm. For Reproductive phase females during lactation, group mean body weight gain was slightly higher than Control for all treated groups. During the Recovery phase, group mean body weight gain for males and females at 12000 ppm during treatment, was higher than Control although group mean body weight on Day R15 for the high dose males and females remained lower than Control. Blood chemistry investigations of Toxicity phase groups revealed dose related increase in urea for males and females and total protein levels for males with statistical significance in males at 12000 ppm. Following recovery, urea levels were comparable to Control in both sexes. Total protein levels in males at 12000 ppm were comparable to Control following a recovery, however total protein levels in females at 12000 ppm were higher than Control with statistical significance . There was a slight dose-related increase in cholesterol levels in both sexes with statistical significance in males at 6000 or 12000 ppm. Following 2-week recovery cholesterol levels at 12000 ppm were comparable to Control. A slight dose-related reduction in phosphorous levels was seen in females at 12000 ppm. Following recovery phosphorous levels remained lower than Control at 12000 ppm for females, without statistical significance. One female at 12000 ppm had an atypically low level. Blood chemistry investigations of Reproductive phase females on Day 13 of lactation showed a statistically significant decreases in creatinine levels in at 12000 ppm, without dose response. There was also a statistically significant increase in triglycerides in females at 12000 ppm, without dose response. Urinalysis investigations performed during Week 5 of treatment revealed dose-related increase in the total protein output in males, with statistical significance at 6000 and 12000 ppm. The protein concentration was also statistically significantly higher than Control in males at 12000 ppm, without dose response. There was a dose-related increase in the total glucose output in males, with statistical significance at 6000 and 12000 ppm. Total glucose output of all female treated groups was higher than Control, without dose response. Glucose concentration was statistically higher than Control in males at 12000 ppm, without dose response. There was a dose-related decrease in total creatinine output and creatinine concentration in males, with statistical significance for creatinine concentration at 6000 and 12000 ppm. Urinalysis was investigated during Week 2 of recovery in both males and females at 12000 ppm, total protein output, protein concentration, total glucose output, glucose concentration, total creatinine output and creatinine concentration were comparable to Control. Following 6 weeks of treatment, there dose-related decreases in terminal body weights for males with statistical significance at 12000 ppm. Terminal body weights for females at 12000 ppm, were low compared to Control with statistical significance. Group mean body weight adjusted liver weights increased in both sexes with statistical significance in all treatment group males and greatest magnitude at 6000 or 12000 ppm. After 2 weeks of recovery, mean adjusted liver weights were comparable to Control at 12000 ppm. A dose-related increase in body weight adjusted kidney weights was observed in the Toxicity phase males with statistical significance at 12000 ppm. Group mean adjusted kidney weights of all treated groups of Toxicity phase females were slightly higher than Control, not to the same extent and with no dose response. After 2 weeks of recovery, group mean adjusted kidney weights for males at 12000 ppm were comparable to Control. Mean adjusted female kidney weights remained higher than Control. Microscopic examination revealed test item-related findings (hyaline droplet accumulation) in the kidneys of all treated Toxicity phase males. Nephropathy and an increase in tubular basophilia, compared to Control, were observed in males at 6000 or 12000 ppm. Microscopic examination performed after 2 weeks of recovery revealed test item-related findings (nephropathy, hyaline droplet accumulation and an increase in tubular basophilia) in the kidneys of several males at 12000 ppm. There were no test item-related findings at microscopic examination for Toxicity phase females.

 

Conclusion:

Dietary administration of test item to Han Wistar rats for 6 weeks at concentrations up to and including 12000 ppm was generally well tolerated by the Reproductive phase females and their offspring. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was 12000 ppm (mean achieved doses; 838 mg/kg bw/day during gestation and 1763 mg/kg bw/day during lactation). Test item-related histopathological changes (nephropathy) in the kidneys of Toxicity phase males at 6000 or 12000 ppm were considered to be adverse, with associated increases in body weight adjusted male kidney weights, a dose-related increase in group mean urea concentration, high total protein concentration in males at 12000 ppm, a dose-related increase in total protein output and total glucose output in the urine, an increase in protein concentration and glucose concentration in the urine of males at 12000 ppm, a dose-related decrease in the total creatinine output and a non-dose-dependent decrease in creatinine concentration in the urine. Hyaline droplets are a common finding in the kidneys of untreated male rats. The increase in hyaline droplets observed in the kidneys of treated males is consistent with a test item associated increase in accumulation of alpha-2u-globulin. Hyaline droplet accumulation due to alpha-2u-globulin is both sex and species specific and is not generally considered to be significant in man. Based on these findings, the NOAEL for general systemic toxicity via the dietary route was concluded to be 2500 ppm (mean achieved dose: 146 mg/kg bw/day) in male rats and 12000 ppm (mean achieved dose: 806 mg/kg bw/day) in female rats.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for specific organ toxicity repeated exposure (STOT-RE).

Since there was no reported significant effects relevant to humans reported at guidance related levels (ORAL ≤ 300 mg/kg bw/day) then there is no requirement to classify STOT-RE.

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017), Section 3.9.2 : Table 3.16 - Equivalent guidance values for 28-day and 90-day studies