Reaction mass of (9E)-9-Undecenal and (9Z)-9-Undecenal and undec-10-enal

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-05-2017 to 23-08-2018

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:

30-05-2017 to 23-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

other: 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 (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males 71 to 78 days old ; females 85 to 92 days old.
- Weight at study initiation: males 328 to 399 g ; females 244 to 327 g. On Day 1 of study all animals were weighed and body weights were reviewed before dosing 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 pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum, removed overnight during the period of urine collection
- Acclimation period: males: eight (8) days before commencement of treatment; females: twenty-two (22) days before commencement of treatment.

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

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

IN-LIFE DATES: From: 2018-01-24 To: 2018-04-17

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for one (1) day at 15-25°C and up to eighteen (18) days at 2-8°C. Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: 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-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.
Results show the formulations to be homogeneous and stable for at least one (1) day at ambient and up to eighteen (18) days refrigerated (2-8°C). Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within the applied limits of ± 10/-15% of the nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during distribution of bottles, magnetic stirring for 2 hours and ambient temperature storage for up to one (1) day and refrigerated storage for up to eighteen (18) days. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (vehicle, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10/-15 % of nominal applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability was confirmed in Corn Oil formulations at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during refrigerated storage in the dark for at least eighteen (18) days.
- Refrigerated formulations were also analysed on day one (1) after preparation, day eight (8) and day eighteen (18): the formulation was removed from storage and equilibrated to ambient temperature. The formulations were mixed according to mixing procedure (as used in the definitive test) by 20-fold inversion followed by magnetic stirring for 20 minutes. Single samples were removed for analysis from the top, middle and bottom of the stirred formulation.
- The analysis consisted of GC FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). The test method was calibrated, with linear regression to calibration standard. Samples of Corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These were then subjected to procedural recovery analysis by GC FID and using internal calibration. The analytical method was validated (details available within the full study report). With LOD = 0.37 μg/mL; linearity = > 0.999 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 5%. Accuracy and precision was confirmed and mean procedural recovery was 101.2 % (n=5 ; CV = 0.75%) at 1 mg/mL and 103.6% (CV = 1.56% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10/-15 % applied limits confirming accurate test item/vehicle formulation.

Duration of treatment / exposure:

Toxicity phase and recovery groups: Two weeks pre-paring after minimum of 6 weeks treatment: as 4 weeks exposure and 2 week recovery period.
Reproductive phase groups: Minimum of two weeks of treatment and two weeks pairing and gestation until day 13 lactation.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1; corn oil vehicle (5 mL/kg applied dose)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

325 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group 4

No. of animals per sex per dose:

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
100 mg/kg bw/day (toxicity test) = 10
325 mg/kg bw/day (toxicity test) = 10
1000 mg/kg bw/day(toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
100 mg/kg bw/day (reproduction test) = 10
100 mg/kg bw/day (toxicity test) = 5
325 mg/kg bw/day (reproduction test) = 10
325 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (reproduction test) = 10
1000 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this study (0, 100, 325 and 1000 mg/kg bw/day) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ± 20% of the mean for the appropriate sex and/or due to poor condition or ill health.
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during acclimatisation at least once daily ; during treatment at least twice daily.

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: See 'Other' below.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded Weekly (including recovery phase). For Toxicity and Recovery phase animals. Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males during Week 4. Food consumption was recorded for females during the periods Days 0-2, 3-6, 7-9, 10-13, 14-16, 17-19 after mating and Days 1-3, 4-6, 7-9 and 10-12 of lactation.

FOOD EFFICIENCY: No.
- 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). Since no significant effects observed no quantitative measurements were performed.
- Time schedule for examinations: Daily (see above).

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Peripheral blood samples: All toxicity groups at termination and recovery groups week 2. For reproductive phase: Day 14 of lactating (females) and test termination (males and females).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight).
- 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).
- 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 6) and all recovery phase (Recovery Week 2)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight).
- Parameters checked: Clarity and Color (App) - by visual assessment, Volume (Vol) - using a measuring cylinder, pH - using a pH meter, Specific gravity (SG), Ketones (Keto), Bile pigments (Bili), Urobilinogen (Urob), Blood pigments (UBld), Protein total output (T-Prot)*, Protein concentration (Prot), Creatinine total output (T-Creat)*, Creatinine concentration (U-Creat), Glucose total output (T-Gluc)*, Glucose concentration (U-Gluc), Sodium (T-Na), Potassium (T-K), Chloride (T-Cl) [where * = derived value) ; Other abnormal components (A)#, Epithelial cells (Epi)#, Leucocytes (WBC)#, Erythrocytes (RBC)#, Casts# (where #= not performed in recovery phase).

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

IMMUNOLOGY: No

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

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

THYROID HORMONE ANALYSIS: Yes
- Time schedule:
(i) at day 4 of age, F1 offspring, two females per litter (where possible) - no females were selected when total litter size dropped below ten/litter or if the resultant number of live females were to be less than 3 for subsequent day 13 procedures. One F1 female for T3/T4 (serum) and One F1 female for TSH (plasma).
(ii) At day 13 of age, F1 offspring, two males and two females per litter (where possible) – one male and one female for T3/T4 (serum) where possible and one male and one female for TSH (plasma) where possible
(iii) at the end of treatment: males from toxicity phase and recovery phase
(iv) at termination: Recovery phase F0 males and all recovery phase F0 females surviving to scheduled termination

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides (left and right), Spleen, Heart, Testes (left and right), Kidneys, Thymus, Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with oviducts)
For reproductive phase females: Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.
For offspring: Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. On day 4: F1 externally abnormal offspring examined and abnormal tissues retained. On day 13: All F1 animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.

HISTOPATHOLOGY: Yes, in five lowest numbered surviving toxicity study males, all toxicity phase females, all recovery phase animals and all adult decedents
- Organs and tissues preserved in neutral buffered 10% formalin or Davidson’s fluid (testes, initially and eyes) as applicable: Abnormalities, Adrenals, Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Epididymides, Eyes, Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from 2 lobes), Lungs (section from two major lobes including bronchi), Lymph nodes - left axillary and mesenteric, Ovaries, Peyer’s Patch, Prostate, Sciatic nerve, Seminal vesicles with coagulating glands, Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical level), Spleen, Sternum (with marrow), Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus with cervix (weighed with oviducts), Vagina
Microscopic analysis was conducted thereof. Any macroscopically observed abnormalities or lesions were also processed. Including in reproductive phase.
- Other: Further information in attached tables.

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.

Data types were analyzed at each timepoint separately (see full study report for full list of data types analyzed):

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

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

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

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

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

Other examinations of gestation, estrous cycles and so forth were examined (Cytel 1995).

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.

Mortality:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain from Day 1 to Day 43 for males/females given the test item was lower than controls, although there was no dose relationship. There was some evidence of recovery during the 15 days off dose. During recovery, body weight gain was unaffected by previous treatment. As such the effects were not considered adverse.

For females in gestation and lactation:
There were no test item related effects on group mean body weight change during gestation and lactation in reproductive phase females. The occasional statistical significances noted at 1000 mg/kg bw/day were considered to reflect normal variation rather than an effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant test item related effects on food consumption of males or females over Weeks 1 to 6 or for females during gestation and lactation.

Food efficiency:

not examined

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

no effects observed

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):

Assessment of haematological parameters in toxicity phase males or females at the end of the treatment period revealed no test item related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Assessment of blood chemistry parameters at the end of the treatment period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Assessment of urinary parameters at the end of the treatment and recovery period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength, Motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

During lactation, forelimb grip strength and group mean total high beam motor activity was statistically significantly lower for females given 1000 mg/kg/day but the values were within historical control data ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Males:
At 1000 mg/kg bw/day, at scheduled termination of the toxicity phase animals after 5 weeks of treatment, male animals given 1000 mg/kg/day had statistically higher body weight adjusted seminal vesicle weights compared to control males (1.24X Control). This difference was not apparent in the recovery phase animals following 2 weeks off dose. It was considered much of this increase was attributed to Male-32 which was heavier bodyweight than other males in the study including recovery phase. Necropsy did not note it enlarged/heavier than normal. As such it was not considered an adverse finding.

There were no further test item related effects on organ weights in males or females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
At 1000 mg/kg bw/day, pale areas of liver were seen in two males given 1000 mg/kg/day. No test item related changes were seen at microscopic examination of the full list of tissues/organs examined; the hepatocyte vacuolation seen in animals given 1000 mg/kg/day and the controls was considered coincidental and as this finding correlated with the macroscopically pale liver in only one male given 1000 mg/kg/day this was considered to be not test item related.

The incidence and distribution of all findings were considered incidental and unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.

Hepatocyte vacuolation was seen in some males and females given 1000 mg/kg/day and some controls. As this finding had similar incidence and/or severity in the controls and treated animals, it was considered as incidental and unrelated to the test item. All other histological findings were considered incidental and unrelated to test item.

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:
Group mean T4 (thyroxine) concentrations in adult males given 1000 mg/kg/day were slightly lower (0.86X Control) than the concurrent control group, but were within the region of the endogenous levels seen in the control matrix used to prepare QC samples. Group mean T4 concentrations in adult males given 100 or 325 mg/kg/day were similar to the controls.

It was considered there were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels.

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 1000 mg/kg bw/day.

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 oral gavage administration for at least five weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received oral gavage test item at doses of 0 (Control), 100, 325, 1000 mg/kg bw/day test item in corn oil vehicle. Ten males were treated in the 100 and 325 mg/kg bw/day doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 1000 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses 0 (Control), 100, 325, 1000 mg/kg bw/day test item in corn oil vehicle. Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks. Recovery phase males were treated for two weeks before pairing up to necropsy after six weeks followed by a 2-week recovery period. Recovery phase females were treated for six weeks followed by a 2-week recovery period. Reproductive phase females were treated for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received the vehicle, corn oil, at the same dose volume as treated groups.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no adverse effect on clinical condition, sensory reactivity, grip strength, motor activity, food consumption, haematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility and gestation length of the adult animals. During lactation, forelimb grip strength was statistically significantly lower for females given 1000 mg/kg/day but the values were within historical control data ranges and is of uncertain relationship to the test item. High beam motor activity during lactation was statistically significantly lower for females given 1000 mg/kg/day, although the response was atypical, with much lower high beam activity levels from minutes 12 to 54 than the control animals. These differences were considered of uncertain relationship to treatment as no similar pattern of behaviour was observed in the Toxicity phase animals. It was considered not to represent an adverse effect of treatment. There were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels.

 

Body weight gain from Day 1 to Day 43 for animals given the test item was lower than controls, although there was no dose relationship. There were no effects on body weight gain during gestation or lactation. The macroscopic examination performed after 5 weeks of treatment revealed pale areas in the liver of two toxicity phase males given 1000 mg/kg/day. No test item related changes were seen at microscopic examination of the full list of tissues/organs examined; the hepatocyte vacuolation seen in animals given 1000 mg/kg/day and the controls was considered coincidental and as this finding correlated with the macroscopically pale liver in only one male given 1000 mg/kg/day this was considered to be not test item related. After five weeks treatment seminal vesicle weights were higher in males given 1000 mg/kg/day however there was no related or otherwise macropathological or micropathological findings. Therefore, the finding was not considered treatment related.

 

Conclusion:

Oral gavage administration of test item to CD rats at concentrations up to and including 1000 mg/kg bw/day was generally well tolerated. There was no adverse effect of treatment on clinical condition, sensory reactivity, motor activity, body weight, food consumption, hematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weights, macropathology or histopathology of the adult animals. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was concluded to be 1000 mg/kg bw/day.

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:

30-05-2017 to 23-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

other: 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 (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males 71 to 78 days old ; females 85 to 92 days old.
- Weight at study initiation: males 328 to 399 g ; females 244 to 327 g. On Day 1 of study all animals were weighed and body weights were reviewed before dosing 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 pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum, removed overnight during the period of urine collection
- Acclimation period: males: eight (8) days before commencement of treatment; females: twenty-two (22) days before commencement of treatment.

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

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

IN-LIFE DATES: From: 2018-01-24 To: 2018-04-17

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

REPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for one (1) day at 15-25°C and up to eighteen (18) days at 2-8°C. Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: 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-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.
Results show the formulations to be homogeneous and stable for at least one (1) day at ambient and up to eighteen (18) days refrigerated (2-8°C). Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within the applied limits of ± 10/-15% of the nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during distribution of bottles, magnetic stirring for 2 hours and ambient temperature storage for up to one (1) day and refrigerated storage for up to eighteen (18) days. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (vehicle, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10/-15 % of nominal applied limits.

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 homogeneity and stability was confirmed in Corn Oil formulations at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during refrigerated storage in the dark for at least eighteen (18) days.
- Refrigerated formulations were also analysed on day one (1) after preparation, day eight (8) and day eighteen (18): the formulation was removed from storage and equilibrated to ambient temperature. The formulations were mixed according to mixing procedure (as used in the definitive test) by 20-fold inversion followed by magnetic stirring for 20 minutes. Single samples were removed for analysis from the top, middle and bottom of the stirred formulation.
- The analysis consisted of GC FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). The test method was calibrated, with linear regression to calibration standard. Samples of Corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These were then subjected to procedural recovery analysis by GC FID and using internal calibration. The analytical method was validated (details available within the full study report). With LOD = 0.37 μg/mL; linearity = > 0.999 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 5%. Accuracy and precision was confirmed and mean procedural recovery was 101.2 % (n=5 ; CV = 0.75%) at 1 mg/mL and 103.6% (CV = 1.56% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10/-15 % applied limits confirming accurate test item/vehicle formulation.

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after six weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 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. 13 weeks. (i.e. after two weeks treatment).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1; corn oil vehicle (5 mL/kg applied dose)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

325 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group 4

No. of animals per sex per dose:

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

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
100 mg/kg bw/day (toxicity test) = 10
325 mg/kg bw/day (toxicity test) = 10
1000 mg/kg bw/day(toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
100 mg/kg bw/day (reproduction test) = 10
100 mg/kg bw/day (toxicity test) = 5
325 mg/kg bw/day (reproduction test) = 10
325 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (reproduction test) = 10
1000 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels selected for investigation in this study (0, 100, 325 and 1000 mg/kg bw/day) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- 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 twice weekly thereafter (including recovery and day of termination). F0 Reproductive phase females: Weekly during acclimatization. Before the feeding of treated diets on the Day that treatment commenced and weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: See 'Other' below.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded Weekly (including recovery phase). For Toxicity and Recovery phase animals. Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males during Week 4. Food consumption was recorded for females during the periods Days 0-2, 3-6, 7-9, 10-13, 14-16, 17-19 after mating and Days 1-3, 4-6, 7-9 and 10-12 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study). Since no significant effects observed no quantitative measurements were performed.
- Time schedule for examinations: Daily (see above).

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 the end of treatment: males from toxicity phase and recovery phase
(iv) at termination: Recovery phase F0 males and all recovery phase F0 females surviving to scheduled termination

Oestrous cyclicity (parental animals):

ESTROUS CYCLE: Yes
- Dry smears – Reproductive females only: taken from 15 days before 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 5 weeks treatment.
- Maternal animals: F0 females failing to produce a viable litter: Day 25 after mating. F0 females: Day 13 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 postmortem 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.

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.

Data types were analyzed at each timepoint separately (see full study report for full list of data types analyzed):

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

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

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

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

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

Other examinations of gestation, estrous cycles and so forth were examined (Cytel 1995).

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:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain from Day 1 to Day 43 for males/females given the test item was lower than controls, although there was no dose relationship. There was some evidence of recovery during the 15 days off dose. During recovery, body weight gain was unaffected by previous treatment. As such the effects were not considered adverse.

For females in gestation and lactation:
There were no test item related effects on group mean body weight change during gestation and lactation in reproductive phase females. The occasional statistical significances noted at 1000 mg/kg bw/day were considered to reflect normal variation rather than an effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant test item related effects on food consumption of males or females over Weeks 1 to 6 or for females during gestation and lactation.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

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):

Assessment of haematological parameters in toxicity phase males or females at the end of the treatment period revealed no test item related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Assessment of blood chemistry parameters at the end of the treatment period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Assessment of urinary parameters at the end of the treatment and recovery period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength, Motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

During lactation, forelimb grip strength and group mean total high beam motor activity was statistically significantly lower for females given 1000 mg/kg/day but the values were within historical control data ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.

Hepatocyte vacuolation was seen in some males and females given 1000 mg/kg/day and some controls. As this finding had similar incidence and/or severity in the controls and treated animals, it was considered as incidental and unrelated to the test item. All other histological findings were considered incidental and unrelated to test item.

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:
Group mean T4 (thyroxine) concentrations in adult males given 1000 mg/kg/day were slightly lower (0.86X Control) than the concurrent control group, but were within the region of the endogenous levels seen in the control matrix used to prepare QC samples. Group mean T4 concentrations in adult males given 100 or 325 mg/kg/day were similar to the controls.

It was considered there were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item related effects on estrous cycle regularity during the 2 weeks prior to pairing. All females showed regular 4-5 day cycles, with the exception of one female in each of the test groups with irregular cycles; these irregular cycles had no impact upon pre-coital interval.

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. There was no effect of treatment on cycle stage at termination.

The group mean number of implantations and litter size was slightly lower than the control in litters from females given 325 or 1000 mg/kg bw/day ; however, as no dose relationship was present and values were within the historical data range, this was considered to be normal biological variation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no 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.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was not affect on fertility or mating performance related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

M10 in litter number 162 (1000 mg/kg/day) was observed to have a swollen mouth on Day 13 of age. This finding correlated with the macroscopic findings for this pup of a soft pale mass attached to the left lower jaw.

No other test item related clinical signs were observed in the offspring.

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, absence of adverse effects to nipple counts and/or only isolated macropathology findings (two instances of squamous cell papillomas) 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.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

With the exception of facial masses in 2 offspring at 1000 mg/kg bw/day, there were no significant macroscopic findings or histopathological finding observed.

At scheduled termination of the offspring two males, from separate litters, within the 1000 mg/kg bw/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papillomas. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental.

Ano-genital distances for male or female offspring were unaffected by treatment with test item. The presence of nipples on Day 13 of age in male offspring from all treated groups was considered not adverse and not test item related as the incidence fell within the historical control ranges and there was no dosage relationship..

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination of the offspring two male pups, from separate litters within the 1000 mg/kg bw/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek. These were confirmed as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental.

There were no other macroscopic findings or histopathological finding observed 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 ANALYSIS
F0 Males:
At 1000 mg/kg bw/day, group mean T4 (thyroxine) concentrations in adult males were slightly lower (0.86X Control) than the concurrent control group, but were within the region of the endogenous levels seen in the control matrix. In lower concentrations T4 concentrations were similar to controls. There was no test item related effects on group mean T3 (triiodothyronine) concentrations.
F1 offspring:
There were no test item related effects on group mean T4 (thyroxine) or T3 (triiodothyronine) concentrations in male or female offspring on Day 13 of age, and group mean concentrations were within the region of the endogenous levels seen in the control matrix.

In the related histopathology:

At scheduled termination of the offspring two male pups, from separate litters within the 1000 mg/kg bw/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek. These were confirmed as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the findings may well be incidental.

There were no other macroscopic findings or histopathological finding observed in the offspring 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. A total of 9 (from 4 litters) at 100 mg/kg bw/day parental treatment, 3 (from 3 litters) at 325 mg/kg bw/day and 8 (from 3 litters) at 1000 mg/kg bw/day male offspring had nipples on Day 13 of age. This incidence of nipple counts in male offspring is within the historical control data range.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 325 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: precautionary NOAEL only, squamous cell papilloma finding was considered to be potentially incidental.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 325 mg/kg bw/day, although for all other parameters the NOAEL was 1000 mg/kg/day. This precautionary NOAEL is on the basis of squamous cell papilloma found in two males at 1000 mg/kg bw/day. It was considered within the study, that there was potential evidence to suggest that the findings may well be incidental.

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 oral gavage administration for at least five weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received oral gavage test item at doses of 0 (Control), 100, 325 or 1000 mg/kg bw/day test item formulated in corn oil vehicle. Ten males were treated in the 100 and 325 mg/kg bw/day doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 (Control) and 1000 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 100, 325 or 1000 mg/kg bw/day. Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks. Recovery phase males were treated for two weeks before pairing up to necropsy after six weeks followed by a 2-week recovery period. Recovery phase females were treated for six weeks followed by a 2-week recovery period. Reproductive phase females were treated for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. Selected F1 offspring were sampled and killed on Day 4 or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age. A similarly constituted Control group was assigned to each phase, and received, the vehicle: corn oil at the same dose volume as treated groups.

 

There were no treatment-related premature mortalities among adult animals during the course of the study. There was no adverse effect on clinical condition, sensory reactivity and grip strength, motor activity, food consumption, haematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility and gestation length on the adult animals. Body weight gain from Day 1 to Day 43 for animals given the test item was lower than controls, although there was no dose relationship. There were no effects on body weight gain during gestation or lactation. During lactation, forelimb grip strength and high beam motor activity was statistically significantly lower for females given 1000 mg/kg bw/day and but this was within the historic control data range or the response was atypical. Macroscopic examination performed after 5 weeks of treatment revealed pale areas in the liver of two toxicity phase males given 1000 mg/kg bw/day. No test item related changes were seen at microscopic examination of the full list of tissues/organs examined; the hepatocyte vacuolation seen in animals given 1000 mg/kg bw/day and the controls was considered coincidental given the finding correlated with the macroscopically pale liver in only one male. After five weeks of treatment, seminal vesicle weights were higher in males given 1000 mg/kg/day. There were no test item related macropathological or micropathological findings, therefore the finding was not considered to be treatment related. There were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring survival, ano-genital distance, body weight or T3 (triiodothyronine) or T4 (thyroxine) levels. The presence of nipples on Day 13 of age in male offspring from all treated groups was considered not adverse and not test item related as the incidence fell within the historical control ranges and there was no dosage relationship. At scheduled termination of the offspring two male pups, from separate litters, within the 1000 mg/kg/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental. There were no other significant macroscopic findings or histopathological finding observed.

 

Conclusion:

Oral gavage administration of test item to CD rats at concentrations up to and including 1000 mg/kg bw/day was generally well tolerated. There was no adverse effect of treatment on clinical condition, sensory reactivity, motor activity, body weight, food consumption, hematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weights, macropathology or histopathology of the adult animals. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring survival, ano-genital distance or body weight. A higher occurrence of nipples were observed in male pups from dams given the test item, but the incidence was within the historical control data range. At scheduled termination of the offspring two male pups, from separate litters, within the 1000 mg/kg/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 325 mg/kg bw/day, although for all other parameters the NOAEL was 1000 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

(9E)-undec-9-enal; (9Z)-undec-9-enal; undec-10-enal

EC Number:

941-821-2

Molecular formula:

C11H20O

IUPAC Name:

(9E)-undec-9-enal; (9Z)-undec-9-enal; undec-10-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Refrigerated (2-8°C) under nitrogen
- Other: Colourless

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males 71 to 78 days old ; females 85 to 92 days old.
- Weight at study initiation: males 328 to 399 g ; females 244 to 327 g. On Day 1 of study all animals were weighed and body weights were reviewed before dosing 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 pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum, removed overnight during the period of urine collection
- Acclimation period: males: eight (8) days before commencement of treatment; females: twenty-two (22) days before commencement of treatment.

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

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

IN-LIFE DATES: From: 2018-01-24 To: 2018-04-17

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

REPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Corn Oil. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for one (1) day at 15-25°C and up to eighteen (18) days at 2-8°C. Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: 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-vehicle formulation: Refrigerated (2 to 8°C), under nitrogen
(ii) Formulated-in vehicle: Refrigerated (2 to 8°C)

- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to eighteen (18) days at 2-8°C and was validated stable for one (1) day at 15-25°C (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. See above and note that the test item was demonstrated to be stable and homogenous when formulated in vehicle.
- 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.
- 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 formulations of test-item/vehicle.

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

OTHER SPECIFICS: Not applicable.
Results show the formulations to be homogeneous and stable for at least one (1) day at ambient and up to eighteen (18) days refrigerated (2-8°C). Formulations were prepared weekly during the treatment period and stored refrigerated (2-8°C).
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within the applied limits of ± 10/-15% of the nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during distribution of bottles, magnetic stirring for 2 hours and ambient temperature storage for up to one (1) day and refrigerated storage for up to eighteen (18) days. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (vehicle, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10/-15 % of nominal applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability was confirmed in Corn Oil formulations at nominal concentrations of 1.00 mg/mL and 200 mg/mL, during refrigerated storage in the dark for at least eighteen (18) days.
- Refrigerated formulations were also analysed on day one (1) after preparation, day eight (8) and day eighteen (18): the formulation was removed from storage and equilibrated to ambient temperature. The formulations were mixed according to mixing procedure (as used in the definitive test) by 20-fold inversion followed by magnetic stirring for 20 minutes. Single samples were removed for analysis from the top, middle and bottom of the stirred formulation.
- The analysis consisted of GC FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). The test method was calibrated, with linear regression to calibration standard. Samples of Corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These were then subjected to procedural recovery analysis by GC FID and using internal calibration. The analytical method was validated (details available within the full study report). With LOD = 0.37 μg/mL; linearity = > 0.999 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 5%. Accuracy and precision was confirmed and mean procedural recovery was 101.2 % (n=5 ; CV = 0.75%) at 1 mg/mL and 103.6% (CV = 1.56% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10/-15 % applied limits confirming accurate test item/vehicle formulation.

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 six weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 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. 13 weeks. (i.e. after two weeks treatment).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1; corn oil vehicle (5 mL/kg applied dose)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Doses / concentrations 3

Dose / conc.:

325 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group 4

No. of animals per sex per dose:

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

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
100 mg/kg bw/day (toxicity test) = 10
325 mg/kg bw/day (toxicity test) = 10
1000 mg/kg bw/day(toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
100 mg/kg bw/day (reproduction test) = 10
100 mg/kg bw/day (toxicity test) = 5
325 mg/kg bw/day (reproduction test) = 10
325 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (reproduction test) = 10
1000 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels selected for investigation in this study (0, 100, 325 and 1000 mg/kg bw/day) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- 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 twice weekly thereafter (including recovery and day of termination). F0 Reproductive phase females: Weekly during acclimatization. Before the feeding of treated diets on the Day that treatment commenced and weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: See 'Other' below.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded Weekly (including recovery phase). For Toxicity and Recovery phase animals. Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males during Week 4. Food consumption was recorded for females during the periods Days 0-2, 3-6, 7-9, 10-13, 14-16, 17-19 after mating and Days 1-3, 4-6, 7-9 and 10-12 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study). Since no significant effects observed no quantitative measurements were performed.
- Time schedule for examinations: Daily (see above).

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 the end of treatment: males from toxicity phase and recovery phase
(iv) at termination: Recovery phase F0 males and all recovery phase F0 females surviving to scheduled termination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No (The requirement for corpora lutea counts is also no longer included in the OECD422 guideline. Therefore this observation has no impact on the regulatory acceptability/compliance of the study).
- 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.

Data types were analyzed at each timepoint separately (see full study report for full list of data types analyzed):

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

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

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

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

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

Other examinations of gestation, estrous cycles and so forth were examined (Cytel 1995).

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:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain from Day 1 to Day 43 for males/females given the test item was lower than controls, although there was no dose relationship. There was some evidence of recovery during the 15 days off dose. During recovery, body weight gain was unaffected by previous treatment. As such the effects were not considered adverse.

For females in gestation and lactation:
There were no test item related effects on group mean body weight change during gestation and lactation in reproductive phase females. The occasional statistical significances noted at 1000 mg/kg bw/day were considered to reflect normal variation rather than an effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant test item related effects on food consumption of males or females over Weeks 1 to 6 or for females during gestation and lactation.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

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):

Assessment of haematological parameters in toxicity phase males or females at the end of the treatment period revealed no test item related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Assessment of blood chemistry parameters at the end of the treatment period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Assessment of urinary parameters at the end of the treatment and recovery period revealed no treatment related effects.

All inter-group differences from controls in the toxicity phase and reproductive phase females at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength, Motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

During lactation, forelimb grip strength and group mean total high beam motor activity was statistically significantly lower for females given 1000 mg/kg/day but the values were within historical control data ranges.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Males:
At 1000 mg/kg bw/day, at scheduled termination of the toxicity phase animals after 5 weeks of treatment, male animals given 1000 mg/kg/day had statistically higher body weight adjusted seminal vesicle weights compared to control males (1.24X Control). This difference was not apparent in the recovery phase animals following 2 weeks off dose. It was considered much of this increase was attributed to Male-32 which was heavier bodyweight than other males in the study including recovery phase. Necropsy did not note it enlarged/heavier than normal. As such it was not considered an adverse finding.

There were no further test item related effects on organ weights in males or females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
At 1000 mg/kg bw/day, pale areas of liver were seen in two males given 1000 mg/kg/day. No test item related changes were seen at microscopic examination of the full list of tissues/organs examined; the hepatocyte vacuolation seen in animals given 1000 mg/kg/day and the controls was considered coincidental and as this finding correlated with the macroscopically pale liver in only one male given 1000 mg/kg/day this was considered to be not test item related.

The incidence and distribution of all findings were considered incidental and unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.

Hepatocyte vacuolation was seen in some males and females given 1000 mg/kg/day and some controls. As this finding had similar incidence and/or severity in the controls and treated animals, it was considered as incidental and unrelated to the test item. All other histological findings were considered incidental and unrelated to test item.

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:

effects observed, non-treatment-related

Description (incidence and severity):

1. Thyroid Hormone Assessment:
Group mean T4 (thyroxine) concentrations in adult males given 1000 mg/kg/day were slightly lower (0.86X Control) than the concurrent control group, but were within the region of the endogenous levels seen in the control matrix used to prepare QC samples. Group mean T4 concentrations in adult males given 100 or 325 mg/kg/day were similar to the controls.

It was considered there were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Group mean number of implantations and litter size was slightly lower than the control in litters from females given 325 or 1000 mg/kg/day; however, as no dose relationship was present and values were within the historical data range, this was considered to be normal biological variation.

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):

All females showed normal estrous cycles before treatment. 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)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Maternal abnormalities

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 effect of parental treatment on offspring body weight

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:

no effects observed

Description (incidence and severity):

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

Group mean number of implantations and litter size was slightly lower than the control in litters from females given 325 or 1000 mg/kg/day; however, as no dose relationship was present and values were within the historical data range, this was considered to be normal biological variation.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination of the offspring two males, from separate litters, within the 1000 mg/kg bw/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papillomas. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental.

Ano-genital distances for male or female offspring were unaffected by treatment with test item. The presence of nipples on Day 13 of age in male offspring from all treated groups was considered not adverse and not test item related as the incidence fell within the historical control ranges and there was no dosage relationship..

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)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 325 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: precautionary NOAEL only, squamous cell papilloma finding was considered to may be incidental.

Remarks on result:

other: squamous cell papilloma finding, which was considered to be potentially incidental - used for precautionary NOAEL setting

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

Description (incidence and severity):

Two male pups, from separate litters, within the 1000 mg/kg/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental.

Overall developmental toxicity

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 was considered to be 1000 mg/kg bw/day. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 325 mg/kg bw/day, although for all other parameters the NOAEL was 1000 mg/kg/day. This precautionary NOAEL is on the basis of squamous cell papilloma found in two males at 1000 mg/kg bw/day. It was considered within the study, that there was potential evidence to suggest that the findings may well be incidental.

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 oral gavage administration for at least five weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received oral gavage test item at doses of 0 (Control), 100, 325 or 1000 mg/kg bw/day test item formulated in corn oil vehicle. Ten males were treated in the 100 and 325 mg/kg bw/day doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 (Control) and 1000 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 100, 325 or 1000 mg/kg bw/day. Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks. Recovery phase males were treated for two weeks before pairing up to necropsy after six weeks followed by a 2-week recovery period. Recovery phase females were treated for six weeks followed by a 2-week recovery period. Reproductive phase females were treated for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. Selected F1 offspring were sampled and killed on Day 4 or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age. A similarly constituted Control group was assigned to each phase, and received, the vehicle: corn oil at the same dose volume as treated groups.

 

There were no treatment-related premature mortalities among adult animals during the course of the study. There was no adverse effect on clinical condition, sensory reactivity and grip strength, motor activity, food consumption, haematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility and gestation length on the adult animals. Body weight gain from Day 1 to Day 43 for animals given the test item was lower than controls, although there was no dose relationship. There were no effects on body weight gain during gestation or lactation. During lactation, forelimb grip strength and high beam motor activity was statistically significantly lower for females given 1000 mg/kg bw/day and but this was within the historic control data range or the response was atypical. Macroscopic examination performed after 5 weeks of treatment revealed pale areas in the liver of two toxicity phase males given 1000 mg/kg bw/day. No test item related changes were seen at microscopic examination of the full list of tissues/organs examined; the hepatocyte vacuolation seen in animals given 1000 mg/kg bw/day and the controls was considered coincidental given the finding correlated with the macroscopically pale liver in only one male. After five weeks of treatment, seminal vesicle weights were higher in males given 1000 mg/kg/day. There were no test item related macropathological or micropathological findings, therefore the finding was not considered to be treatment related. There were no test item related effects on T3 (triiodothyronine) or T4 (thyroxine) levels. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring survival, ano-genital distance, body weight or T3 (triiodothyronine) or T4 (thyroxine) levels. The presence of nipples on Day 13 of age in male offspring from all treated groups was considered not adverse and not test item related as the incidence fell within the historical control ranges and there was no dosage relationship. At scheduled termination of the offspring two male pups, from separate litters, within the 1000 mg/kg/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental. There were no other significant macroscopic findings or histopathological finding observed.

 

Conclusion:

Oral gavage administration of test item to CD rats at concentrations up to and including 1000 mg/kg bw/day was generally well tolerated. There was no adverse effect of treatment on clinical condition, sensory reactivity, motor activity, body weight, food consumption, hematology, blood chemistry, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weights, macropathology or histopathology of the adult animals. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day. There was no effect of parental treatment on offspring clinical signs, sex ratio, offspring survival, ano-genital distance or body weight. A higher occurrence of nipples were observed in male pups from dams given the test item, but the incidence was within the historical control data range. At scheduled termination of the offspring two male pups, from separate litters, within the 1000 mg/kg/day group, were found to have soft pale masses, one on the left lower jaw and one on the left inner cheek, confirmed histologically as squamous cell papilloma. A relationship to treatment is uncertain. It was considered within the study, that there was potential evidence to suggest that the test item may well be incidental. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 325 mg/kg bw/day, although for all other parameters the NOAEL was 1000 mg/kg/day.