(1RS,4Z,8E,12RS)-13-oxabicyclo[10.1.0]trideca-4,8-diene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-04-2016 to 20-03-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-04-2016 to 20-03-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: At ambient temperature in the dark
- Other: colourless to pale yellow

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: At ambient temperature in the dark
(ii) Formulated-diet: Formulations were prepared weekly and frozen in aliquots to allow daily feeding. Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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:

Sprague-Dawley

Remarks:

Crl:CD(SD)

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 (full details reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males ca. 71 days old ; females ca. 85 days old.
- Weight at study initiation: males 312 to 383 g ; females 229 to 299 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 ; (removed overnight before blood sampling for hematology, blood chemistry, urine collection and adult thyroid hormone investigations.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: six days before commencement of treatment; females: twenty 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: 2016-09-28 To: 2016-12-23

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 ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL dissolved into acetonee, 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 = 3.078 μg/mL and LOQ = 10.261 μg/mL; linearity = > 0.995 between 5 μg/mL and 25 μg/mL. Repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 93.78% (n=5 ; CV = 0.71%) at 100 ppm and 99.9% (CV = 1.95; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 30 hours at 100 ppm and 2 days at 20000 ppm. At frozen temperature (-10 to -30°C) for up to 40 days. Frozen
stability at 20000 ppm was not assessed in error, however degradation and formulation instability is more pronounced at lower levels. As frozen stability was confirmed at 100 ppm for 40 days and all subsequent testing of achieved concentration samples were well within acceptable limits. It is considered that there is sufficient evidence to conclude test diets with a concentration of 20000 ppm are stable for 40 days under frozen conditions.
- Mean concentrations of diet-formulations analysed during the study were within ± 10/15% 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 5% 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 6 weeks exposure and 2 week recovery period.
Reproductive phase groups: Minimum of two weeks of treatment and two weeks pairing and gestation until day 14 lactation.

Frequency of treatment:

Continuous via diet

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet

Dose / conc.:

417 ppm

Remarks:

Low – Group 2

Dose / conc.:

1 250 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

3 750 ppm

Remarks:

High – Group 4

Dose / conc.:

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

Remarks:

Overall Mean for males for 417 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for males for 1250 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for males for 3750 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for females for 417 ppm (Main, Toxicity and Recovery phase groups)

Dose / conc.:

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

Remarks:

Overall Mean for females for 1250 ppm (Main, Toxicity and Recovery phase groups)

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm (Main, Toxicity and Recovery phase groups)

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm during 6 weeks (Toxicity and Recovery phase animals and Main phase females for two weeks pre-pairing).

No. of animals per sex per dose:

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
417 ppm (toxicity test) = 10
1250 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 5
3750 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
417 ppm (reproduction test) = 10
417 ppm (toxicity test) = 5
1250 ppm (reproduction test) = 10
1250 ppm (toxicity test) = 5
3750 ppm (reproduction test) = 10
3750 ppm (toxicity test) = 5
3750 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, 417, 1250 and 3750 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), 2500, 5000 and 7500 ppm respectively. It was determined that 7500 ppm would be unsuitable for a subsequent toxicity study. The overall mean achieved doses in animals receiving 2500, 5000 or 7500 ppm were 144, 304 and 470 mg/kg/day in males and 147, 322 and 490 mg/kg/day in females, respectively. There were no mortalities and no clinical signs related to treatment noted. No clear dose-related effect on food consumption or bodyweight gain was observed in males. Females receiving 7500 ppm initially lost weight and took 14 days to regain parity with their pre-treatment body weight. At 5000 ppm the females showed only 49% of the weight gain of the Control group over the 14-day exposure period, whilst at 2500 ppm the animals gained 86% of the Control weight gain. A non-dose related reduction in food consumption was seen in the females with a greater indication of a slight effect on food intake seen in animals receiving 5000 ppm than in animals receiving 7500 ppm. It is likely that these effects on body weight and food consumption were due to the reduced palatability of the formulated diets that was manifested in the females but not in the males. It was considered that this dietary concentration would be unsuitable for use in a study of longer duration. Water consumption was unaffected by treatment. There were no macroscopic findings that were considered to be related to treatment. High absolute and body weight adjusted liver weights were seen in males and females given 7500 ppm but with no macroscopic correlations.
- 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 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 (week 3) but recommenced for males in week 4. 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: No, although eyes were subject to pathology procedures at necropsy
- Time schedule for examinations: At termination only.
- 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: Yes (overnight, no more than 20 hours fasting)
- 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: Yes (overnight, no more than 20 hours fasting)
- 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 #= may not be 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 termination: All F0 males and females in toxicity phase and recovery phase

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.

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 analysed 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 analysed 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:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment related mortalities.

Group 1 male 36 was found dead during Week 7 of treatment. Prior to demise there had been no signs at routine physical examination and the individual was in good general condition. No macroscopic abnormalities were seen at necropsy. Following microscopic examination, no findings were seen that would have contributed to demise. Within the study the demise was not considered test item related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment.

Females:
Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Achieved doses generally maintained the intervals between dietary concentrations and increased during lactation due to increased physiological demand.

Males:
An initial reduction in food intake was observed in 3750 ppm group of main study and recovery phase males were 92% of control (89% for main study and toxicity/recovery phase females). Following completion of treatment food consumption. For males that previously received 3750 ppm was similar to concurrent Controls

Females:
An initial reduction in food intake was observed in 3750 ppm group main study and toxicity/recovery phase females pf 89% of control. Following completion of treatment females that previously received 3750 ppm food consumption was high when compared with Controls; approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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:

not examined

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 revealed no treatment related changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were minor biochemical changes in blood plasma but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
At 3750 ppm: after 6 weeks of treatment males showed slightly elevated total protein. Males at both 1250 and 3750 ppm concentrations demonstrated low plasma bile acid concentration but without dose response. These findings were not evident in toxicity phase females after 6 weeks of treatment or in main study (reproductive) females on Day 14 of lactation. After two weeks without treatment dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls.
Females:
No effects.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were minor biochemical changes in urine but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
After 6 weeks of treatment males at all dose levels showed low urinary specific gravity
At 3750 ppm: within males demonstrated increased urinary output when compared with Controls
Females:
At 3750 ppm: females demonstrated high urinary pH with Controls

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and hindlimb grip strength, motor activity scores and arena observations appeared unaffected by treatment.

Any observations appeared isolated, or when with inter-group statistical significance: without dose-response and/or within control ranges and/or historic control ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 3750 ppm: after 6 weeks of treatment males showed low adjusted mean heart weight (p<0.05) and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). There were no correlating micropathological/histopathological findings to account these differences compared to Controls. In addition the adjusted epididymal and glans penis weights were low but these differences are not considered to be of biological significance. After two weeks of recovery adjusted epididymal, glans penis, heart, adrenal and thyroid weights for males that received 3750 ppm were not significantly different from Controls.
Thymus weights after two weeks of recovery for males that received 3750 ppm were significantly low when compared with Controls; in the absence of an effect after 6 weeks of treatment or in the recovery phase females this is considered not to be of toxicological significance.

Females:
At 3750 ppm: after 6 weeks of treatment toxicity phase females showed significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; i.e. 108% of controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; i.e. 115% of controls).
On Day 14 of lactation females at 3750 ppm had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls.

See: “histopathology : non-neoplastic” for further comments, regarding lack of biological significance and/or relevance to man.

Gross pathological findings:

effects observed, non-treatment-related

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.

On Day 14 of lactation pale areas were seen on the liver of one female at 1250 ppm and one female at 3750 ppm. In addition depressions were seen in the corpus region of the stomach of one female at 417 ppm and one female at 3750 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males (m21 and m25) and in one female (f104) previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively.
THYROIDS: follicular cell hypertrophy was seen in males (m22 and m23) and females treated, and in a few males treated with 417 or 1250 ppm. This hypertrophy contributed to the increased body weight adjusted group mean combined thyroid and parathyroid weights in males treated with 3750 ppm, compared with male controls. Following the 2 week recovery period, follicular cell hypertrophy was seen in two males previously treated with 3750 ppm for 6 weeks. Therefore, in males, the relevant incidence in main study and recovery phase animals was the same (40%) and so recovery had not occurred but complete recovery was seen in females.

Females:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males and in one female previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively. It was noted the hypertrophy at the end of treatment and recovery would have contributed to the increased body weight adjusted group mean liver weights in females that were treated with 3750 ppm. Examination of the liver obtained from females on Day 14 of lactation showed centrilobular hepatocyte hypertrophy in the majority of females treated with 1250 or 3750ppm and in one female treated with 417 ppm and exhibited a dose-response relationship.
THYROIDS: follicular cell hypertrophy was seen in males and females treated with complete recovery seen.

Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction as the rat. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man.

Incidental Findings
THYMUS : minimal or slight involution/atrophy was seen in a few control females and females treated with 3750 ppm. As this change was not seen in any unmated control females or females treated with 3750 ppm for 6 weeks, it was considered that the involution/atrophy was related to the ‘stress’ of pregnancy and lactation within rodents and therefore unrelated to treatment (literature citations given in the full study report). The involution/atrophy was considered to have contributed to the recorded decreased body weight adjusted group mean thymus weights in females treated with 3750 ppm, compared with female controls.

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:

>= 3 750 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 223 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 750 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:

>= 225 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

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males is considered to be 3750 ppm which was equivalent to actual dose received: 223 mg/kg body weight per day. The no-observed-adverse-effect level (NOAEL) for females is considered to be 3750 ppm which was equivalent to actual dose received: 225 mg/kg bw/day. The test item related histopathological changes in the kidneys of males, were considered within the study to be non-adverse (and/or would be adaptive changes). Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, It was considered that this was a species specific observation and is not toxicologically 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 six 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 or ten female rats which received dietary test item at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Five females were treated in the 0 (control) and 3750 ppm doses for toxicity but not used pairing purposes with the reproductive phase males. Recovery phase groups included five males and females treated at 0 ppm (Control) and 3750 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Main study males were treated for two weeks before pairing up to necropsy after at least six weeks. Toxicity phase females were treated for at least six weeks. Main study males were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 offspring received no direct administration of the test item; any exposure was in utero or via the milk prior to termination. A similarly constituted Control group was assigned to each F0 phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 417, 1250 or 3750 ppm for the toxicity and recovery phases were 25.1, 75.0 and 223 mg/kg bw/day in males and 0 and 232 mg/kg bw/day in 0 (control) and 3750 ppm females, respectively. Mean achieved doses at 417, 1250 or 3750 ppm for the main study (reproductive phase), toxicity and recovery females was: 26.9, 75.6 and 225 mg/kg bw/day. The mean achieved dose during gestation in main study females was: 28.7, 87.0 and 259 mg/kg bw/day and during lactation was: 67.3, 192 and 598 mg/kg bw/day. Mean achieved doses were higher due to the increased physiological demand of lactation.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no treatment-related effect on clinical condition, sensory reactivity and (hind/fore limb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. There was no effect on water intake, 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, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary. During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm indicated body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males at 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery). Males indicated food consumption that was similar to concurrent Controls. Blood chemistry investigations after 6 weeks of treatment males at 3750 ppm demonstrated slightly elevated total protein and at 1250 and 3750 ppm, low plasma bile acid concentration without dose-response. These findings were not evident in toxicity phase females or in main study females on Day 14 of lactation. After two weeks recovery the total protein, albumin concentration and albumin/globulin ratio for recovery phase males at 3750 ppm was comparable with Controls. Recovery was therefore evident. Urinalysis investigations performed after 6 weeks of treatment to males at all dose levels indicated low urinary specific gravity, whereas at 3750 ppm males had increased urinary output and females high urinary pH when compared with Control values. After two weeks of recovery males at 3750 ppm had urinary output and specific gravity that were essentially comparable with Controls.Males that received 3750 ppm had low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery for males : adjusted heart, adrenal and thyroid weights were not significantly different from Controls. Toxicity phase females at 3750 ppm had significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; approximately 115% of Controls). At 3750 ppm, on Day 14 of lactation females had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls. Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

 

Conclusion:

Dietary administration of test item to Sprague-Dawly rats for 6 weeks at concentrations up to and including 3750 ppm was generally well tolerated by the Toxicity phase males/females, and 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 systemic toxicity was 3750 ppm (mean achieved dose males/females 223 mg/kg bw/day) and/or reproductive/developmental toxicity at 3750 ppm (mean achieved doses females; 259 mg/kg bw/day during gestation and 598 mg/kg bw/day during lactation). Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man. Based on these findings, the NOAEL for general systemic toxicity via the dietary route was concluded to be 3750 ppm (mean achieved dose: 223 mg/kg bw/day) in male/female rats.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-04-2016 to 20-03-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: At ambient temperature in the dark
- Other: colourless to pale yellow

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: At ambient temperature in the dark
(ii) Formulated-diet: Formulations were prepared weekly and frozen in aliquots to allow daily feeding. Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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:

Sprague-Dawley

Remarks:

Crl:CD(SD)

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 (full details reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males ca. 71 days old ; females ca. 85 days old.
- Weight at study initiation: males 312 to 383 g ; females 229 to 299 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 ; (removed overnight before blood sampling for hematology, blood chemistry, urine collection and adult thyroid hormone investigations.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: six days before commencement of treatment; females: twenty 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: 2016-09-28 To: 2016-12-23

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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 ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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.

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: No

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 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL dissolved into acetonee, 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 = 3.078 μg/mL and LOQ = 10.261 μg/mL; linearity = > 0.995 between 5 μg/mL and 25 μg/mL. Repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 93.78% (n=5 ; CV = 0.71%) at 100 ppm and 99.9% (CV = 1.95; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 30 hours at 100 ppm and 2 days at 20000 ppm. At frozen temperature (-10 to -30°C) for up to 40 days. Frozen
stability at 20000 ppm was not assessed in error, however degradation and formulation instability is more pronounced at lower levels. As frozen stability was confirmed at 100 ppm for 40 days and all subsequent testing of achieved concentration samples were well within acceptable limits. It is considered that there is sufficient evidence to conclude test diets with a concentration of 20000 ppm are stable for 40 days under frozen conditions.
- Mean concentrations of diet-formulations analysed during the study were within ± 10/15% 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 5% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 4%, confirming precise analysis.

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of six weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 14 of lactation.

Frequency of treatment:

Daily; at approximately the same time each day.
F1 generation were not dosed.

Details on study schedule:

- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 14 weeks. (i.e. after two weeks treatment).

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet

Dose / conc.:

417 ppm

Remarks:

Low – Group 2

Dose / conc.:

1 250 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

3 750 ppm

Remarks:

High – Group 4

Dose / conc.:

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

Remarks:

Overall Mean for males for 417 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for males for 1250 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for males for 3750 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for females for 417 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Dose / conc.:

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

Remarks:

Overall Mean for females for 1250 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm during 6 weeks (Toxicity and Recovery phase animals and Main phase females for two weeks pre-pairing).

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 417 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 1250 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 3750 ppm group

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 417 ppm group ; mean achieved doses were higher due to the increased physiological demand of lactation

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 1250 ppm group ; mean achieved doses were higher due to the increased physiological demand of lactation

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 3750 ppm group ; mean achieved doses were higher due to the increased physiological demand of lactation

No. of animals per sex per dose:

10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 417, 1250 and 3750 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), 2500, 5000 and 7500 ppm respectively. It was determined that 7500 ppm would be unsuitable for a subsequent toxicity study. The overall mean achieved doses in animals receiving 2500, 5000 or 7500 ppm were 144, 304 and 470 mg/kg/day in males and 147, 322 and 490 mg/kg/day in females, respectively. There were no mortalities and no clinical signs related to treatment noted. No clear dose-related effect on food consumption or bodyweight gain was observed in males. Females receiving 7500 ppm initially lost weight and took 14 days to regain parity with their pre-treatment body weight. At 5000 ppm the females showed only 49% of the weight gain of the Control group over the 14-day exposure period, whilst at 2500 ppm the animals gained 86% of the Control weight gain. A non-dose related reduction in food consumption was seen in the females with a greater indication of a slight effect on food intake seen in animals receiving 5000 ppm than in animals receiving 7500 ppm. It is likely that these effects on body weight and food consumption were due to the reduced palatability of the formulated diets that was manifested in the females but not in the males. It was considered that this dietary concentration would be unsuitable for use in a study of longer duration. Water consumption was unaffected by treatment. There were no macroscopic findings that were considered to be related to treatment. High absolute and body weight adjusted liver weights were seen in males and females given 7500 ppm but with no macroscopic correlations.
- 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

Parental animals: Observations and examinations:

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 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 (week 3) but recommenced for males in week 4. Reproductive females after mating food consumption was recorded daily until Day 13 of lactation.

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

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. 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 termination: All F0 males and females in toxicity phase and recovery phase

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
testis weight, epididymis weight and histology/histopathology
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae count in male offspring , macroscopic pathology / abnormalities. Histopathology. Thyroid Hormone Analysis.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 Males: After 6 weeks treatment.
- Maternal animals: F0 females failing to produce a viable litter: Day 25 after mating. F0 females: Day 14 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues were prepared for microscopic examination and weighed, respectively. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed at scheduled intervals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to post-mortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Particular attention was paid to the external genitalia. Thyroids were retained from one male and one female offspring in each litter on Day 13 of age.

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid tissues were prepared for microscopic examination from one male and one female offspring in each litter. Organ weights were not performed for F1 males or F1 females. Any abnormal tissues were retained in an appropriate fixative.

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 analysed 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.

Reproductive indices:

Mating performance, fertility, gestation length, gestation index

Offspring viability indices:

Post-implantation survival index, viability index, lactation index and sex ratio

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:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment related mortalities.

Group 1 male 36 was found dead during Week 7 of treatment. Prior to demise there had been no signs at routine physical examination and the individual was in good general condition. No macroscopic abnormalities were seen at necropsy. Following microscopic examination, no findings were seen that would have contributed to demise. Within the study the demise was not considered test item related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment.

Females:
Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Achieved doses generally maintained the intervals between dietary concentrations and increased during lactation due to increased physiological demand.

Males:
An initial reduction in food intake was observed in 3750 ppm group of main study and recovery phase males were 92% of control (89% for main study and toxicity/recovery phase females). Following completion of treatment food consumption. For males that previously received 3750 ppm was similar to concurrent Controls

Females:
An initial reduction in food intake was observed in 3750 ppm group main study and toxicity/recovery phase females pf 89% of control. Following completion of treatment females that previously received 3750 ppm food consumption was high when compared with Controls; approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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:

not examined

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 revealed no treatment related changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were minor biochemical changes in blood plasma but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
At 3750 ppm: after 6 weeks of treatment males showed slightly elevated total protein. Males at both 1250 and 3750 ppm concentrations demonstrated low plasma bile acid concentration but without dose response. These findings were not evident in toxicity phase females after 6 weeks of treatment or in main study (reproductive) females on Day 14 of lactation. After two weeks without treatment dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls.
Females:
No effects.

Urinalysis findings:

no effects observed

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):

no effects observed

Description (incidence and severity):

Sensory reactivity and hindlimb grip strength, motor activity scores and arena observations appeared unaffected by treatment.

Any observations appeared isolated, or when with inter-group statistical significance: without dose-response and/or within control ranges and/or historic control ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males (m21 and m25) and in one female (f104) previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively.
THYROIDS: follicular cell hypertrophy was seen in males (m22 and m23) and females treated, and in a few males treated with 417 or 1250 ppm. This hypertrophy contributed to the increased body weight adjusted group mean combined thyroid and parathyroid weights in males treated with 3750 ppm, compared with male controls. Following the 2 week recovery period, follicular cell hypertrophy was seen in two males previously treated with 3750 ppm for 6 weeks. Therefore, in males, the relevant incidence in main study and recovery phase animals was the same (40%) and so recovery had not occurred but complete recovery was seen in females.

Females:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males and in one female previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively. It was noted the hypertrophy at the end of treatment and recovery would have contributed to the increased body weight adjusted group mean liver weights in females that were treated with 3750 ppm. Examination of the liver obtained from females on Day 14 of lactation showed centrilobular hepatocyte hypertrophy in the majority of females treated with 1250 or 3750ppm and in one female treated with 417 ppm and exhibited a dose-response relationship.
THYROIDS: follicular cell hypertrophy was seen in males and females treated with complete recovery seen.

Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction as the rat. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man.

Incidental Findings
THYMUS : minimal or slight involution/atrophy was seen in a few control females and females treated with 3750 ppm. As this change was not seen in any unmated control females or females treated with 3750 ppm for 6 weeks, it was considered that the involution/atrophy was related to the ‘stress’ of pregnancy and lactation within rodents and therefore unrelated to treatment (literature citations given in the full study report). The involution/atrophy was considered to have contributed to the recorded decreased body weight adjusted group mean thymus weights in females treated with 3750 ppm, compared with female controls.

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the Main study (Reproductive phase) and toxicity and recovery groups of the study showed regular 4 or 5 days estrous cycles prior to the start of treatment (during acclimatisation).

The majority of main study females showed regular 4/5 day estrous cycles during the treatment period prior to mating; one Control female showed an irregular cycle and one female at 3750 ppm was acyclic. Pre-coital interval for main study females was unaffected by treatment with all females, including the acyclic female at 3750 ppm, mating at the first estrus after pairing.

Examination of vaginal smears prior to termination of the Reproductive, Toxicity and Recovery phase females did not reveal any adverse effects of treatment.

There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days. The gestation index was 100% in all groups. All toxicity and recovery phase females showed estrus before termination and all reproductive phase females were in diestrus.

Estrous cycles, pre-coital interval, mating performance, fertility and gestation length/index were unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no toxicologically significant treatment related findings. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

All other histological changes were considered to be unrelated to 6 weeks of treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no affect on fertility or mating performance related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 750 ppm

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 223 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 750 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:

>= 225 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

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs related to treatment within F1 males or females.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no treatment related mortality associated with treatment within F1 males or females. This is considering that there was no effect of parental treatment on litter size, offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, nipple counts or macropathology in offspring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no treatment related effect on body weight associated with treatment within F1 males or females.

On Day 1 of age the mean body weights for both male and female offspring derived from groups treated with test item were essentially similar to Control and considered unaffected by parental treatment. Subsequent weight gain up to Day 7 of age was similar to Controls. However, at 1250 and 3750 ppm between Days 7 and 13 of age body weight gain for male and female offspring were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm (p<0.01) and for females at both 1250 ppm (p<0.05) and 3750 ppm (p<0.01); a dose response was apparent. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm (p<0.05) and for female offspring at 1250ppm (p<0.05) and 3750ppm (p<0.05). This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distances for male or female offspring were unaffected by treatment with test item.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Male nipple counts were considered unaffected by treatment with test item.

A few male offspring were observed with nipples on Day 13 of age, however the distribution across the groups showed no relationship to treatment.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings or histopathological finding observed in the offspring that died prior to scheduled termination or at scheduled termination on Day 13 of age that were attributable to parental treatment with the test item..

On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat, which may be related to a slight delay in development because of the slightly larger mean litter size at this dose level ; there were no other findings that could be related to
parental treatment.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings or histopathological finding observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment with the test item.

See 'Other Effects' for Thyroid Hormone Analysis and related Histopathology

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.

In the related histopathology:

There were no macroscopic findings or histopathological finding observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment with the test item.

2. Offspring Ano-Genital Distance and Male Nipple Counts
Ano-genital distances for male or female offspring were unaffected by treatment with test item. Male nipple counts were unaffected by treatment with test item.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 750 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

gestation

Generation:

F1

Effect level:

>= 259 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

lactation

Generation:

F1

Effect level:

>= 598 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in male/female rats was 3750 ppm equivalent to actual dose received: 223 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for reproductive / developmental toxicity is considered to be 3750 ppm, which was equivalent to actual dose received: 259 mg/kg bw/day (gestation) and 598 mg/kg bw/day (lactation).
No reproductive / developmental toxicity effects related to the test item were observed in parental males/females or offspring during the study.

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 six 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 or ten female rats which received dietary test item at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Five females were treated in the 0 (control) and 3750 ppm doses for toxicity but not used pairing purposes with the reproductive phase males. Recovery phase groups included five males and females treated at 0 ppm (Control) and 3750 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Main study males were treated for two weeks before pairing up to necropsy after at least six weeks. Toxicity phase females were treated for at least six weeks. Main study males were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 offspring received no direct administration of the test item; any exposure was in utero or via the milk prior to termination. A similarly constituted Control group was assigned to each F0 phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 417, 1250 or 3750 ppm for the toxicity and recovery phases were 25.1, 75.0 and 223 mg/kg bw/day in males and 0 and 232 mg/kg bw/day in 0 (control) and 3750 ppm females, respectively. Mean achieved doses at 417, 1250 or 3750 ppm for the main study (reproductive phase), toxicity and recovery females was: 26.9, 75.6 and 225 mg/kg bw/day. The mean achieved dose during gestation in main study females was: 28.7, 87.0 and 259 mg/kg bw/day and during lactation was: 67.3, 192 and 598 mg/kg bw/day. Mean achieved doses were higher due to the increased physiological demand of lactation.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no treatment-related effect on clinical condition, sensory reactivity and (hind/fore limb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. There was no effect on water intake, 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, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary. During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm indicated body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males at 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery). Males indicated food consumption that was similar to concurrent Controls. Blood chemistry investigations after 6 weeks of treatment males at 3750 ppm demonstrated slightly elevated total protein and at 1250 and 3750 ppm, low plasma bile acid concentration without dose-response. These findings were not evident in toxicity phase females or in main study females on Day 14 of lactation. After two weeks recovery the total protein, albumin concentration and albumin/globulin ratio for recovery phase males at 3750 ppm was comparable with Controls. Recovery was therefore evident. Urinalysis investigations performed after 6 weeks of treatment to males at all dose levels indicated low urinary specific gravity, whereas at 3750 ppm males had increased urinary output and females high urinary pH when compared with Control values. After two weeks of recovery males at 3750 ppm had urinary output and specific gravity that were essentially comparable with Controls. Males that received 3750 ppm had low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery for males : adjusted heart, adrenal and thyroid weights were not significantly different from Controls. Toxicity phase females at 3750 ppm had significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; approximately 115% of Controls). At 3750 ppm, on Day 14 of lactation females had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls. Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

 

Within F1 offspring, the clinical condition, litter size, sex ratio and offspring survival were unaffected by parental treatment. Ano-genital distance of both males and females on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. On Day 1 of age the mean body weights for both male and female offspring derived from groups receiving the test item were essentially similar to Control and considered unaffected by parental treatment. Subsequent weight gain up to Day 7 of age was similar to Controls. However between Days 7 and 13 of age body weight gain for male and female offspring at 1250 and 3750 ppm were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm and for females at 1250 ppm and 3750 ppm. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm and for female offspring at 1250ppm and 3750ppm. This was associated with slightly larger litter sizes in these groups. Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings related to treatment. On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat, which may be related to a slight delay in development because of the slightly larger mean litter size at this dose level. Therefore, not considered an effect of treatment.

 

Conclusion:

Dietary administration of test item to Sprague-Dawley rats for 6 weeks at concentrations up to and including 3750 ppm was generally well tolerated by the Toxicity phase males/females, and 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 systemic toxicity was 3750 ppm (mean achieved dose males/females 223 mg/kg bw/day) and/or reproductive/developmental toxicity at 3750 ppm (mean achieved doses females; 259 mg/kg bw/day during gestation and 598 mg/kg bw/day during lactation). The changes in the thyroid glands observed in rodents were consequences of hepatic enzyme induction were non-adverse (adaptive effects) and/or are of little toxicological relevance to man. Reproductive performance, fertility and offspring survival were unaffected by parental treatment. From Day 7 of age growth was slightly reduced for offspring in the groups receiving 1250 or 3750 ppm; this was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment. Based on these findings, the NOAEL for general systemic toxicity and reproductive/developmental toxicity via the dietary route was concluded to be 3750 ppm.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 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: December 2016 ; signature: March 2017

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

EC Number:

945-746-6

Molecular formula:

Not Applicable

IUPAC Name:

1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: At ambient temperature in the dark
- Other: colourless to pale yellow

Specific details on test material used for the study:

- Physical state: Liquid
- Storage condition of test material: At ambient temperature in the dark
- Other: colourless to pale yellow

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: At ambient temperature in the dark
(ii) Formulated-diet: Formulations were prepared weekly and frozen in aliquots to allow daily feeding. Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (full details reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males ca. 71 days old ; females ca. 85 days old.
- Weight at study initiation: males 312 to 383 g ; females 229 to 299 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 ; (removed overnight before blood sampling for hematology, blood chemistry, urine collection and adult thyroid hormone investigations.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: six days before commencement of treatment; females: twenty 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: 2016-09-28 To: 2016-12-23

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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 ; 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 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 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). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- 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 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL dissolved into acetonee, 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 = 3.078 μg/mL and LOQ = 10.261 μg/mL; linearity = > 0.995 between 5 μg/mL and 25 μg/mL. Repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 93.78% (n=5 ; CV = 0.71%) at 100 ppm and 99.9% (CV = 1.95; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 30 hours at 100 ppm and 2 days at 20000 ppm. At frozen temperature (-10 to -30°C) for up to 40 days. Frozen
stability at 20000 ppm was not assessed in error, however degradation and formulation instability is more pronounced at lower levels. As frozen stability was confirmed at 100 ppm for 40 days and all subsequent testing of achieved concentration samples were well within acceptable limits. It is considered that there is sufficient evidence to conclude test diets with a concentration of 20000 ppm are stable for 40 days under frozen conditions.
- Mean concentrations of diet-formulations analysed during the study were within ± 10/15% 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 5% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 4%, confirming precise analysis.

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of six weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 14 of lactation.

Frequency of treatment:

Daily; at approximately the same time each day.
F1 generation were not dosed.

Duration of test:

- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 14 weeks. (i.e. after two weeks treatment).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet

Doses / concentrations 2

Dose / conc.:

417 ppm

Remarks:

Low – Group 2

Doses / concentrations 3

Dose / conc.:

1 250 ppm

Remarks:

Intermediate – Group 3

Doses / concentrations 4

Dose / conc.:

3 750 ppm

Remarks:

High – Group 4

Doses / concentrations 5

Dose / conc.:

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

Remarks:

Overall Mean for males for 417 ppm group

Doses / concentrations 6

Dose / conc.:

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

Remarks:

Overall Mean for males for 1250 ppm group

Doses / concentrations 7

Dose / conc.:

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

Remarks:

Overall Mean for males for 3750 ppm group

Doses / concentrations 8

Dose / conc.:

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

Remarks:

Overall Mean for females for 417 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Doses / concentrations 9

Dose / conc.:

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

Remarks:

Overall Mean for females for 1250 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Doses / concentrations 10

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm (Main, Toxicity and Recovery phase groups during the two week pre-pairing treatment period)

Doses / concentrations 11

Dose / conc.:

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

Remarks:

Overall Mean for females for 3750 ppm during 6 weeks (Toxicity and Recovery phase animals and Main phase females for two weeks pre-pairing).

Doses / concentrations 12

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 417 ppm group

Doses / concentrations 13

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 1250 ppm group

Doses / concentrations 14

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (gestation) for 3750 ppm group

Doses / concentrations 15

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 417 ppm group ; mean achieved
doses were higher due to the increased physiological demand of lactation

Doses / concentrations 16

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 1250 ppm group ; mean achieved
doses were higher due to the increased physiological demand of lactation

Doses / concentrations 17

Dose / conc.:

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

Remarks:

Overall Mean for reproductive phase females (lactation) for 3750 ppm group ; mean achieved
doses were higher due to the increased physiological demand of lactation

No. of animals per sex per dose:

10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 417, 1250 and 3750 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), 2500, 5000 and 7500 ppm respectively. It was determined that 7500 ppm would be unsuitable for a subsequent toxicity study. The overall mean achieved doses in animals receiving 2500, 5000 or 7500 ppm were 144, 304 and 470 mg/kg/day in males and 147, 322 and 490 mg/kg/day in females, respectively. There were no mortalities and no clinical signs related to treatment noted. No clear dose-related effect on food consumption or bodyweight gain was observed in males. Females receiving 7500 ppm initially lost weight and took 14 days to regain parity with their pre-treatment body weight. At 5000 ppm the females showed only 49% of the weight gain of the Control group over the 14-day exposure period, whilst at 2500 ppm the animals gained 86% of the Control weight gain. A non-dose related reduction in food consumption was seen in the females with a greater indication of a slight effect on food intake seen in animals receiving 5000 ppm than in animals receiving 7500 ppm. It is likely that these effects on body weight and food consumption were due to the reduced palatability of the formulated diets that was manifested in the females but not in the males. It was considered that this dietary concentration would be unsuitable for use in a study of longer duration. Water consumption was unaffected by treatment. There were no macroscopic findings that were considered to be related to treatment. High absolute and body weight adjusted liver weights were seen in males and females given 7500 ppm but with no macroscopic correlations.
- 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

Examinations

Maternal examinations:

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 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 (week 3) but recommenced for males in week 4. Reproductive females after mating food consumption was recorded daily until Day 13 of lactation.

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

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. 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 termination: All F0 males and females in toxicity phase and recovery phase

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

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 analysed 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.

Indices:

Offspring viability indices including; post-implantation survival index, live birth index, viability index, lactation index and sex ratio.

Historical control data:

Historical control data was available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment related mortalities.

Group 1 male 36 was found dead during Week 7 of treatment. Prior to demise there had been no signs at routine physical examination and the individual was in good general condition. No macroscopic abnormalities were seen at necropsy. Following microscopic examination, no findings were seen that would have contributed to demise. Within the study the demise was not considered test item related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment.

Females:
Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

AAchieved doses generally maintained the intervals between dietary concentrations and increased during lactation due to increased physiological demand.

Males:
An initial reduction in food intake was observed in 3750 ppm group of main study and recovery phase males were 92% of control (89% for main study and toxicity/recovery phase females). Following completion of treatment food consumption. For males that previously received 3750 ppm was similar to concurrent Controls

Females:
An initial reduction in food intake was observed in 3750 ppm group main study and toxicity/recovery phase females pf 89% of control. Following completion of treatment females that previously received 3750 ppm food consumption was high when compared with Controls; approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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:

not examined

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 revealed no treatment related changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were minor biochemical changes in blood plasma but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
At 3750 ppm: after 6 weeks of treatment males showed slightly elevated total protein. Males at both 1250 and 3750 ppm concentrations demonstrated low plasma bile acid concentration but without dose response. These findings were not evident in toxicity phase females after 6 weeks of treatment or in main study (reproductive) females on Day 14 of lactation. After two weeks without treatment dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls.
Females:
No effects.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were minor biochemical changes in urine but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
After 6 weeks of treatment males at all dose levels showed low urinary specific gravity
At 3750 ppm: within males demonstrated increased urinary output when compared with Controls
Females:
At 3750 ppm: females demonstrated high urinary pH with Controls

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and hindlimb grip strength, motor activity scores and arena observations appeared unaffected by treatment.

Any observations appeared isolated, or when with inter-group statistical significance: without dose-response and/or within control ranges and/or historic control ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 3750 ppm: after 6 weeks of treatment males showed low adjusted mean heart weight (p<0.05) and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). There were no correlating micropathological/histopathological findings to account these differences compared to Controls. In addition the adjusted epididymal and glans penis weights were low but these differences are not considered to be of biological significance. After two weeks of recovery adjusted epididymal, glans penis, heart, adrenal and thyroid weights for males that received 3750 ppm were not significantly different from Controls.
Thymus weights after two weeks of recovery for males that received 3750 ppm were significantly low when compared with Controls; in the absence of an effect after 6 weeks of treatment or in the recovery phase females this is considered not to be of toxicological significance.

Females:
At 3750 ppm: after 6 weeks of treatment toxicity phase females showed significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; i.e. 108% of controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; i.e. 115% of controls).
On Day 14 of lactation females at 3750 ppm had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls.

See: “histopathology : non-neoplastic” for further comments, regarding lack of biological significance and/or relevance to man.

Gross pathological findings:

effects observed, non-treatment-related

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.

On Day 14 of lactation pale areas were seen on the liver of one female at 1250 ppm and one female at 3750 ppm. In addition depressions were seen in the corpus region of the stomach of one female at 417 ppm and one female at 3750 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males (m21 and m25) and in one female (f104) previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively.
THYROIDS: follicular cell hypertrophy was seen in males (m22 and m23) and females treated, and in a few males treated with 417 or 1250 ppm. This hypertrophy contributed to the increased body weight adjusted group mean combined thyroid and parathyroid weights in males treated with 3750 ppm, compared with male controls. Following the 2 week recovery period, follicular cell hypertrophy was seen in two males previously treated with 3750 ppm for 6 weeks. Therefore, in males, the relevant incidence in main study and recovery phase animals was the same (40%) and so recovery had not occurred but complete recovery was seen in females.

Females:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males and in one female previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively. It was noted the hypertrophy at the end of treatment and recovery would have contributed to the increased body weight adjusted group mean liver weights in females that were treated with 3750 ppm. Examination of the liver obtained from females on Day 14 of lactation showed centrilobular hepatocyte hypertrophy in the majority of females treated with 1250 or 3750ppm and in one female treated with 417 ppm and exhibited a dose-response relationship.
THYROIDS: follicular cell hypertrophy was seen in males and females treated with complete recovery seen.

Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction as the rat. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man.

Incidental Findings
THYMUS : minimal or slight involution/atrophy was seen in a few control females and females treated with 3750 ppm. As this change was not seen in any unmated control females or females treated with 3750 ppm for 6 weeks, it was considered that the involution/atrophy was related to the ‘stress’ of pregnancy and lactation within rodents and therefore unrelated to treatment (literature citations given in the full study report). The involution/atrophy was considered to have contributed to the recorded decreased body weight adjusted group mean thymus weights in females treated with 3750 ppm, compared with female controls.

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.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

It was considered offspring survival was not affected by treatment.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter size, offspring survival was not affected by treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation length and gestation index were unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The majority of main study females showed regular 4/5 day estrous cycles during the treatment period prior to mating; one Control female showed an irregular cycle and one female at 3750 ppm was acyclic. Pre-coital interval for main study females was unaffected by treatment with all females, including the acyclic female at 3750 ppm, mating at the first estrus after pairing. It was considered estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. Litter size, offspring survival was not affected by treatment.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 750 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 259 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment related Adverse Effects observed up to the maximum dose level

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no treatment related effect of parental treatment on offspring body weight.

On Day 1 of age the mean body weights for both male and female offspring derived from groups treated with test item were essentially similar to Control and considered unaffected by parental treatment.

Subsequent weight gain up to Day 7 of age was similar to Controls. However, at 1250 and 3750 ppm between Days 7 and 13 of age body weight gain for male and female offspring were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm (p<0.01) and for females at both 1250 ppm (p<0.05) and 3750 ppm (p<0.01); a dose response was apparent. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm (p<0.05) and for female offspring at 1250ppm (p<0.05) and 3750ppm (p<0.05). This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring survival

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring sex ratio

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of parental treatment on offspring litter size or offspring body weight

On Day 1 of age the mean body weights for both male and female offspring derived from groups treated with test item were essentially similar to Control and considered unaffected by parental treatment.

Subsequent weight gain up to Day 7 of age was similar to Controls. However, at 1250 and 3750 ppm between Days 7 and 13 of age body weight gain for male and female offspring were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm (p<0.01) and for females at both 1250 ppm (p<0.05) and 3750 ppm (p<0.01); a dose response was apparent. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm (p<0.05) and for female offspring at 1250ppm (p<0.05) and 3750ppm (p<0.05). This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Ano-genital distances for male or female offspring were unaffected by treatment with test item.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Other effects:

no effects observed

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 3 750 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

gestation

Effect level:

>= 259 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Key result

Dose descriptor:

NOAEL

Remarks:

lactation

Effect level:

>= 598 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in male/female rats was 3750 ppm equivalent to actual dose received: 223 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for reproductive / developmental toxicity is considered to be 3750 ppm, which was equivalent to actual dose received: 259 mg/kg bw/day (gestation) and 598 mg/kg bw/day (lactation).
No reproductive / developmental toxicity effects related to the test item were observed in parental males/females or offspring during the study.

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 six 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 or ten female rats which received dietary test item at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Five females were treated in the 0 (control) and 3750 ppm doses for toxicity but not used pairing purposes with the reproductive phase males. Recovery phase groups included five males and females treated at 0 ppm (Control) and 3750 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Main study males were treated for two weeks before pairing up to necropsy after at least six weeks. Toxicity phase females were treated for at least six weeks. Main study males were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 offspring received no direct administration of the test item; any exposure was in utero or via the milk prior to termination. A similarly constituted Control group was assigned to each F0 phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 417, 1250 or 3750 ppm for the toxicity and recovery phases were 25.1, 75.0 and 223 mg/kg bw/day in males and 0 and 232 mg/kg bw/day in 0 (control) and 3750 ppm females, respectively. Mean achieved doses at 417, 1250 or 3750 ppm for the main study (reproductive phase), toxicity and recovery females was: 26.9, 75.6 and 225 mg/kg bw/day. The mean achieved dose during gestation in main study females was: 28.7, 87.0 and 259 mg/kg bw/day and during lactation was: 67.3, 192 and 598 mg/kg bw/day. Mean achieved doses were higher due to the increased physiological demand of lactation.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no treatment-related effect on clinical condition, sensory reactivity and (hind/fore limb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. There was no effect on water intake, 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, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary. During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm indicated body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males at 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery). Males indicated food consumption that was similar to concurrent Controls. Blood chemistry investigations after 6 weeks of treatment males at 3750 ppm demonstrated slightly elevated total protein and at 1250 and 3750 ppm, low plasma bile acid concentration without dose-response. These findings were not evident in toxicity phase females or in main study females on Day 14 of lactation. After two weeks recovery the total protein, albumin concentration and albumin/globulin ratio for recovery phase males at 3750 ppm was comparable with Controls. Recovery was therefore evident. Urinalysis investigations performed after 6 weeks of treatment to males at all dose levels indicated low urinary specific gravity, whereas at 3750 ppm males had increased urinary output and females high urinary pH when compared with Control values. After two weeks of recovery males at 3750 ppm had urinary output and specific gravity that were essentially comparable with Controls. Males that received 3750 ppm had low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery for males : adjusted heart, adrenal and thyroid weights were not significantly different from Controls. Toxicity phase females at 3750 ppm had significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; approximately 115% of Controls). At 3750 ppm, on Day 14 of lactation females had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls. Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

 

Within F1 offspring, the clinical condition, litter size, sex ratio and offspring survival were unaffected by parental treatment. Ano-genital distance of both males and females on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. On Day 1 of age the mean body weights for both male and female offspring derived from groups receiving the test item were essentially similar to Control and considered unaffected by parental treatment. Subsequent weight gain up to Day 7 of age was similar to Controls. However between Days 7 and 13 of age body weight gain for male and female offspring at 1250 and 3750 ppm were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm and for females at 1250 ppm and 3750 ppm. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm and for female offspring at 1250ppm and 3750ppm. This was associated with slightly larger litter sizes in these groups. Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings related to treatment. On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat, which may be related to a slight delay in development because of the slightly larger mean litter size at this dose level. Therefore, not considered an effect of treatment.

 

Conclusion:

Dietary administration of test item to Sprague-Dawley rats for 6 weeks at concentrations up to and including 3750 ppm was generally well tolerated by the Toxicity phase males/females, and 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 systemic toxicity was 3750 ppm (mean achieved dose males/females 223 mg/kg bw/day) and/or reproductive/developmental toxicity at 3750 ppm (mean achieved doses females; 259 mg/kg bw/day during gestation and 598 mg/kg bw/day during lactation). The changes in the thyroid glands observed in rodents were consequences of hepatic enzyme induction were non-adverse (adaptive effects) and/or are of little toxicological relevance to man. Reproductive performance, fertility and offspring survival were unaffected by parental treatment. From Day 7 of age growth was slightly reduced for offspring in the groups receiving 1250 or 3750 ppm; this was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment. Based on these findings, the NOAEL for general systemic toxicity and reproductive/developmental toxicity via the dietary route was concluded to be 3750 ppm.