cis-2-methyl-4-propyl-1,3-oxathiane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-10-2017 to 02-05-2018

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:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-10-2017 to 02-05-2018

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: January 2018 ; signature: June 2018

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), in the dark under nitrogen and under yellow light when handled.
(ii) Formulated: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre-vehicle formulation: Not applicable.
(ii) Formulated: In a preceding (1) formulation and method validation and (2) prior/within definitive testing: the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was demonstrated for up to 15 days following refrigerated storage (2 to 8°C) and for 21 days following ambient storage (15 to 25°C). Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test item. In the dose formulation analysis, mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: The test item was found to be adequately soluble in corn oil vehicle formulations up to 200 mg/mL. The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Procedural recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. The mean concentrations of test item in formulations analysed for the study were within 10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.
- 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. For each concentration, approximately 40% of the required volume of corn oil was added to the required amount of test item, and magnetically stirred until uniformly mixed. The remaining vehicle was added, and the formulation magnetically stirred until homogeneous.
- 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.

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 70 to 77 days old ; females 84 to 91 days old.
- Weight at study initiation: males 344 to 418 g ; females 242 to 308 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: seven days before commencement of treatment; females: 21 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: 2018-02-28 To: 2018-05-09

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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 twenty-one (21) day at 15 to 25°C and up to fifteen (15) days at 2 to 8°C. Formulations were prepared weekly during the treatment period and stored at ambient (15 to 25°C), in amber glass bottles.

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: 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. Results show the formulations to be homogeneous and stable in vehicle.

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: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to fifteen (15) days at 2-8°C and was validated stable for twenty-one (21) days 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.

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: The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. This can be excluded as per laboratory SOP. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 21 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 10% of the initial time zero value and the coefficient of variation was less than 2%.
- The analysis consisted of GC-FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). Samples in corn oil were accurately fortified with known amounts of test item in an end solution in teradecane. These were then subjected to analysis by GC-FID analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). With LOD = 0.0499 μg/mL; linearity = > 0.99 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 1%. Accuracy and precision was confirmed and mean procedural recovery was 97.6% (n=5 ; CV = 1.79%) at 2 mg/mL and 99.3% (CV = 0.89% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% 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.:

150 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

600 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
150 mg/kg bw/day (toxicity test) = 10
300 mg/kg bw/day (toxicity test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
150 mg/kg bw/day (reproduction test) = 10
150 mg/kg bw/day (toxicity test) = 5
300 mg/kg bw/day (reproduction test) = 10
300 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (reproduction test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a previously conducted 14-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 14-day sighting test were: Group 1: control (vehicle: Corn oil), Group 2: 300 mg/kg bw/day, Group 3: 600 mg/kg bw/day and group 4: 1000 mg/kg bw/day. In the 14-day sighting test (administered consecutively, for 14-days) the following effects were determined. Group 4 (high dose: 1000 mg/kg bw/day) was terminated early due to marked toxicity (reduced body tone and uncoordinated gait). There were no clinical signs in the 300 or 600 mg/kg bw/day dose levels. At 600 mg/kg bw/day, males had slightly lower overall body weight gain. Males/females at 600 mg/kg bw/day and females only at 300 mg/kg bw/day had periods of slightly lower food intake but overall food consumption was unaffected. In both males and females, liver weights were higher with a dose relationship at 300 and 600 mg/kg bw/day. There were no adverse macroscopic findings. Basis: other: nominal in vehicle (corn oil)
- 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). Water consumption was recorded daily by weight for each cage of animals, using water bottles fitted with sipper tubes from the end of Week 2. Water consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males during Week 4. Water consumption was recorded continuously for Toxicity phase and Recovery phase females.
- 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 that were considered adverse and related to treatment.

At 600 mg/kg bw/day: one male had piloerection on day 20, and transient salivation was evident following dosing in week 5 in four males. Transient salivation, with or without chin rubbing, was occasionally seen in females during gestation at 150, 300 and 600 mg/kg bw/day. There was no adverse effect on sensory reactivity and grip strength, motor activity.
For females in gestation and lactation:
Transient salivation, with or without chin rubbing, was occasionally seen during gestation at 150, 300 or 600 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities considered directly related to treatment.

One male treated at 300 mg/kg/day (M-18) was humanely terminated on day 22 for poor condition. Subject to necropsy. Microscopic examination revealed vacuolation of the periportal hepatocytes that was identified in animals terminated following five weeks of treatment. One female (F-58) experienced mortality during blood sampling on day 13. There were no relevant microscopic or macroscopic findings. This was not considered treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: body weight gain was variable throughout the treatment period in males and non-mated females. Overall gains were similar to control in both.
Females:
No significant findings. In non-mated males body weight gain was variable throughout the treatment period.

For females in gestation and lactation:
At 150, 300 or 600 mg/kg bw/day: body weight gain was variable but overall unaffected by treatment. During lactation, body weight gain was variable, low in days 4-7 and high in days 7-13. Overall bodyweight gain was comparable to Control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in males. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.
Females:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in non-paired females. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.

For females in gestation and lactation:
At 150, 300 and 600 mg/kg bw/day: food consumption was mostly similar to controls, with exception of 600 mg/kg bw/day where it was low during days 17 to 20. Food intact was low during day 4-10 lactation, but overall was considered unaffected.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in males. Over the full recovery period it was considered unaffected by treatment.
Females:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in non mated females. Over the full recovery period it was considered low.

For females in gestation and lactation:
Water consumption was consistently high during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic changes.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in haematological parameters that would be considered adverse.

Examinations following two-week recovery and on day 13 of lactation did not identify any differences to control that were attributable to treatment. All differences to controls were considered minor or did not show dose-response relationships and therefore were considered the result of normal biological variation

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in blood chemistry parameters that would be considered adverse.

At 600 mg/kg bw/day: there was elevated plasma cholesterol concentration in non-mated females and elevated plasma urea concentration in males. It was considered these differences may be associated with test item related microscopic changes in the male kidney and the liver of both sexes at the corresponding dose level. Plasma urea concentration remained high in males following two-weeks without treatment.

See ‘Gross pathological findings’ and ‘histopathological findings – non-neoplastic’ for further information

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was higher but differences failed to show a dose response.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Females:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was lower.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Following two-week recovery and/or at Day 13 lactation: differences to controls were considered minor or the result of 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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all males ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of all males. Hyaline droplets were confirmed to be composed of α2μ globulin (immunohistochemistry). All males were graded as having minimal to slight α2μ globulin present.
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of three males
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
Females:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all females
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation

All other findings were considered unrelated to treatment.

Dose-dependent hepatocellular periportal vacuolation in males and females correlated at 600 mg/kg bw/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5, respectively. Intracellular lipids accumulate when their rate of degradation no longer exceeds their rate of synthesis or their release into the bloodstream is slowed (see citations in the full study report). The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg bw/day. Examination of the liver in males and females, following two weeks without treatment revealed partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes. Therefore the changes were likely not to be adverse. In addition, a dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg bw/day. Hyaline droplets are composed of poorly catabolized α2μ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The higher incidence and multifocal nature of the basophilic tubules seen in this group most likely reflects damage and is adverse Examination of the kidneys from Control and males previously treated at 600 mg/kg bw/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Although basophilic tubules were seen in two Control males and one male receiving 150 mg/kg bw/day of the test item they were only present focally and were consistent with background levels.

Toxicity to humans is considered unlikely as little or no α2μ globulin is present in humans.

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:
At 300 and 600 mg/kg bw/day: T4 concentration in blood T4 concentration in blood from males treated for five weeks was statistically significantly lower than Control.
At 150 mg/kg bw/day: T4 concentration in blood from males was marginally lower than Control.
T4 concentrations in males treated at 600 mg/kg bw/day were comparable to Control and endogenous levels following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.
T4 concentrations in blood from male and female offspring were comparable with endogenous levels.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Prior to treatment all females had regular estrus cycles of 4 to 5 days. All females had a regular estrus cycle of 4 to 5 days during the treatment period, with the exception of one at 600 mg/kg bw/day that had an irregular cycle, one at 600 mg/kg bw/day that had an extended estrus, and two at 300 mg/kg bw/day and one at 600 mg/kg bw/day that showed acyclicity. All non-mated females showed regular estrous cycles at the end of treatment and during the two week period of no treatment at 600 mg/kg bw/day.

There was no effect of treatment on pre-coital interval, mating performance, gestation length and gestation index. Two females receiving 600 mg/kg bw/day that showed evidence of mating, failed to litter.

All females were in diestrus at termination on Day 13 of lactation.

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:

not specified

Description (incidence and severity):

There was no effect of treatment on pre-coital interval, mating performance, gestation length and gestation index. Two females receiving 600 mg/kg bw/day that showed evidence of mating, failed to litter.

At 600 mg/kg bw/day, two females were not pregnant and three litters died by Day 2 of age that was attributed to the reduced secretory activity of the mammary gland in F1 females. Two of these females exhibited irregularities of the estrous cycle before mating. Of the three females with litter loss, one showed extended estrus, and a second had an irregular cycle.

The offspring from adults treated at 600 mg/kg bw/day were small on Day 1 of age and subsequent growth, especially during Days 4-7, to Day 13 was poor and correlated with clinical signs of poor maternal care.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no in-life clinical signs reported in offspring, following parental treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Two females receiving 600 mg/kg bw/day were not pregnant and three litters died by Day 2 of lactation; the maternal mammary glands of these females showed reduced secretory activity. Some offspring at 300 or 600 mg/kg bw/day had clinical signs that indicated poor maternal care.

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):

There were no significant macroscopic findings or histopathological finding observed.

Ano-genital distances for male or female offspring were unaffected by treatment with test item. Male offspring did not develop nipples.

Decedent F1 offspring had no milk in stomach and autolysis of stomach contents. There was perianal scabs on some members of liters at 300 and 600 mg/kg bw/day which was indicative of poor maternal care.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no significant histopathological findings 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 300 and 600 mg/kg bw/day: T4 concentration in blood T4 concentration in blood from males treated for five weeks was statistically significantly lower than Control.
At 150 mg/kg bw/day: T4 concentration in blood from males was marginally lower than Control.
T4 concentrations in males treated at 600 mg/kg bw/day were comparable to Control and endogenous levels following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.
F1 offspring:
T4 concentrations in blood from male and female offspring were comparable with endogenous levels.

2. Offspring Ano-Genital Distance and Male Nipple Counts
Ano-genital distances for male or female offspring were unaffected by treatment with test item. Male offspring did not develop nipples.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Which correlated with clinical signs of poor maternal care.

Remarks on result:

other: precautionary NOAEL only. due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Which correlated with clinical signs of poor maternal care.

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 600 mg/kg bw/day. The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Applicant assessment indicates: that whilst the effects on lipid metabolism where not adverse in adults since they were deemed reversible, they may have led to adverse outcome in offspring via reduced secretory activity in female adults and consequential poor maternal care. On this basis a precautionary NOAEL has been adopted.

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), 150, 300 or 600 mg/kg bw/day test item formulated in corn oil vehicle. Ten males were treated in the 150 and 300 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 600 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 150, 300 or 600 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, ophthalmic changes and/or pre-coital interval, mating performance gestation length on the adult animals. Body weight gain was variable throughout the treatment period in males and non-mated females at 600 mg/kg bw/day. Overall gains were similar to control in both. Following cessation of treatment, females showed body weight loss, compared with body weight stasis in the Control. Body weight gain was variable during both gestation and lactation, but again overall gains in treated animals were similar to Control. Food consumption was similarly affected by treatment in all study phases, but was noticeably low during Days 4-10 of lactation. Water consumption was consistently high in males and non mated females at 300 or 600 mg/kg bw/day, during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day. There were no toxicologically significant changes in haematology, blood chemistry and plasma parameters following five weeks of treatment in males and unmated females and on Day 13 of lactation except for minor elevated plasma cholesterol concentration in non-mated females and plasma urea concentration in males and minor change in creatinine concentrations in males/females receiving 600 mg/kg bw/day. These changes were attributed to changes in microscopic changes observed in male kidney and/or male/female liver. At the end of treatment liver weight and kidney weight were high in males/females at 300 and 600 mg/kg bw/day and 600 mg/kg bw/day, respectively. Urinalysis indicated a high urinary volume as a result of the higher water intake in males/females at 600 mg/kg bw/day. This may be attributed to higher plasma and urinary protein concentrations. Macroscopic examination performed after 5 weeks of treatment or 2 weeks recovery or reproductive females after day 13 lactation revealed no test item treatment related lesions. Microscopic examination revealed a dose-dependent hepatocellular periportal vacuolation in males and females treated at 150, 300 or 600 mg/kg/day that correlated at 600 mg/kg/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5. This metabolic alteration may also be reflected by the presence of an increase in urinary ketones in all males at 600 mg/kg/day. The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg/day. Examination of the liver in males and females at 600 mg/kg/day, following two weeks without treatment indicated partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes, therefore changes were considered unlikely to be adverse. A dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg/day. Hyaline droplets are composed of poorly catabolized α2µ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The presence of increased α2µ globulin in males was confirmed immunohistochemically. Examination of the kidneys from Control and males previously treated at 600 mg/kg/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Basophilic tubules were only present focally and were consistent with background levels of this finding. Only partial recovery was evident following two weeks without treatment. Relevance for toxicity in humans through this α2µ globulin mechanism is considered unlikely.

 

At 600 mg/kg/day, two females were not pregnant. Three litters indicated mortality by Day 2, that was attributed to the reduced secretory activity of the mammary gland in the adults. Two females exhibited irregularities of the estrous cycle before mating. Of the three females with total litter loss, one showed extended estrus, and a second had an irregular cycle. The offspring from adults treated at 600 mg/kg/day were small on Day 1 of age and subsequent growth, especially during Days 4-7, to Day 13, correlated with clinical signs of poor maternal care. There was some evidence of poor maternal care at 300 mg/kg/day. Ano genital distance in the offspring was unaffected by maternal treatment. Male offspring did not develop nipples. T4 (thyroxine) levels were statistically low in males at 300 or 600 mg/kg bw/day but were comparable with control following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.

 

Conclusion:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 600 mg/kg bw/day. The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Applicant assessment indicates: that whilst the effects on lipid metabolism where not adverse in adults since they were deemed reversible, they may have led to adverse outcome in offspring via reduced secretory activity in female adults and consequential poor maternal care. On this basis a precautionary NOAEL has been adopted.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-10-2017 to 02-05-2018

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:

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: January 2018 ; signature: June 2018

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), in the dark under nitrogen and under yellow light when handled.
(ii) Formulated: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre-vehicle formulation: Not applicable.
(ii) Formulated: In a preceding (1) formulation and method validation and (2) prior/within definitive testing: the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was demonstrated for up to 15 days following refrigerated storage (2 to 8°C) and for 21 days following ambient storage (15 to 25°C). Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test item. In the dose formulation analysis, mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: The test item was found to be adequately soluble in corn oil vehicle formulations up to 200 mg/mL. The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Procedural recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. The mean concentrations of test item in formulations analysed for the study were within 10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.
- 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. For each concentration, approximately 40% of the required volume of corn oil was added to the required amount of test item, and magnetically stirred until uniformly mixed. The remaining vehicle was added, and the formulation magnetically stirred until homogeneous.
- 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.

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

OTHER SPECIFICS: Not applicable.

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 70 to 77 days old ; females 84 to 91 days old.
- Weight at study initiation: males 344 to 418 g ; females 242 to 308 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: seven days before commencement of treatment; females: 21 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: 2018-02-28 To: 2018-05-09

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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 twenty-one (21) day at 15 to 25°C and up to fifteen (15) days at 2 to 8°C. Formulations were prepared weekly during the treatment period and stored at ambient (15 to 25°C), in amber glass bottles.

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: 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. Results show the formulations to be homogeneous and stable in vehicle.

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: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to fifteen (15) days at 2-8°C and was validated stable for twenty-one (21) days 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability: The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. This can be excluded as per laboratory SOP. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 21 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 10% of the initial time zero value and the coefficient of variation was less than 2%.
- The analysis consisted of GC-FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). Samples in corn oil were accurately fortified with known amounts of test item in an end solution in teradecane. These were then subjected to analysis by GC-FID analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). With LOD = 0.0499 μg/mL; linearity = > 0.99 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 1%. Accuracy and precision was confirmed and mean procedural recovery was 97.6% (n=5 ; CV = 1.79%) at 2 mg/mL and 99.3% (CV = 0.89% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% 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).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

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

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

600 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
150 mg/kg bw/day (toxicity test) = 10
300 mg/kg bw/day (toxicity test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
150 mg/kg bw/day (reproduction test) = 10
150 mg/kg bw/day (toxicity test) = 5
300 mg/kg bw/day (reproduction test) = 10
300 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (reproduction test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a previously conducted 14-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 14-day sighting test were: Group 1: control (vehicle: Corn oil), Group 2: 300 mg/kg bw/day, Group 3: 600 mg/kg bw/day and group 4: 1000 mg/kg bw/day. In the 14-day sighting test (administered consecutively, for 14-days) the following effects were determined. Group 4 (high dose: 1000 mg/kg bw/day) was terminated early due to marked toxicity (reduced body tone and uncoordinated gait). There were no clinical signs in the 300 or 600 mg/kg bw/day dose levels. At 600 mg/kg bw/day, males had slightly lower overall body weight gain. Males/females at 600 mg/kg bw/day and females only at 300 mg/kg bw/day had periods of slightly lower food intake but overall food consumption was unaffected. In both males and females, liver weights were higher with a dose relationship at 300 and 600 mg/kg bw/day. There were no adverse macroscopic findings. Basis: other: nominal in vehicle (corn oil)
- 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

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). Water consumption was recorded daily by weight for each cage of animals, using water bottles fitted with sipper tubes from the end of Week 2. Water consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males during Week 4. Water consumption was recorded continuously for Toxicity phase and Recovery phase females.
- 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: Yes (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.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs observed that were considered adverse and related to treatment.

At 600 mg/kg bw/day: one male had piloerection on day 20, and transient salivation was evident following dosing in week 5 in four males. Transient salivation, with or without chin rubbing, was occasionally seen in females during gestation at 150, 300 and 600 mg/kg bw/day. There was no adverse effect on sensory reactivity and grip strength, motor activity.
For females in gestation and lactation:
Transient salivation, with or without chin rubbing, was occasionally seen during gestation at 150, 300 or 600 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities considered directly related to treatment.

One male treated at 300 mg/kg/day (M-18) was humanely terminated on day 22 for poor condition. Subject to necropsy. Microscopic examination revealed vacuolation of the periportal hepatocytes that was identified in animals terminated following five weeks of treatment. One female (F-58) experienced mortality during blood sampling on day 13. There were no relevant microscopic or macroscopic findings. This was not considered treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: body weight gain was variable throughout the treatment period in males and non-mated females. Overall gains were similar to control in both.
Females:
No significant findings. In non-mated males body weight gain was variable throughout the treatment period.

For females in gestation and lactation:
At 150, 300 or 600 mg/kg bw/day: body weight gain was variable but overall unaffected by treatment. During lactation, body weight gain was variable, low in days 4-7 and high in days 7-13. Overall bodyweight gain was comparable to Control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in males. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.
Females:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in non-paired females. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.
For females in gestation and lactation:
At 150, 300 and 600 mg/kg bw/day: food consumption was mostly similar to controls, with exception of 600 mg/kg bw/day where it was low during days 17 to 20. Food intact was low during day 4-10 lactation, but overall was considered unaffected.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in males. Over the full recovery period it was considered unaffected by treatment.
Females:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in non mated females. Over the full recovery period it was considered low.

For females in gestation and lactation:
Water consumption was consistently high during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic changes.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in haematological parameters that would be considered adverse.

Examinations following two-week recovery and on day 13 of lactation did not identify any differences to control that were attributable to treatment. All differences to controls were considered minor or did not show dose-response relationships and therefore were considered the result of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

AThere were no toxicologically significant changes in blood chemistry parameters that would be considered adverse.

At 600 mg/kg bw/day: there was elevated plasma cholesterol concentration in non-mated females and elevated plasma urea concentration in males. It was considered these differences may be associated with test item related microscopic changes in the male kidney and the liver of both sexes at the corresponding dose level. Plasma urea concentration remained high in males following two-weeks without treatment.

See ‘Gross pathological findings’ and ‘histopathological findings – non-neoplastic’ for further information

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was higher but differences failed to show a dose response.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Females:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was lower.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Following two-week recovery and/or at Day 13 lactation: differences to controls were considered minor or the result of 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:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: liver weight and kidney weight were high
At 300 mg/kg bw/day: liver weight were high
Females:
At 600 mg/kg bw/day: liver weight and kidney weight were high
At 300 mg/kg bw/day: liver weight were high
At 150 mg/kg bw/day: liver weight were high
All other findings were considered minor or the result of normal biological variation.

Adjusted liver and kidney weights remained high, although less markedly, in males and females previously treated at 600 mg/kg/day when compared with Control. Absolute and adjusted organ weights were unaffected by treatment on Day 13 of lactation.

See ‘Gross pathological findings’ and ‘histopathological findings – non-neoplastic’ for further information.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination performed after 5 weeks of treatment or 2 weeks recovery or reproductive females after day 13 lactation revealed no test item treatment related lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all males ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of all males. Hyaline droplets were confirmed to be composed of α2μ globulin (immunohistochemistry). All males were graded as having minimal to slight α2μ globulin present.
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of three males
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
Females:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all females
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation

All other findings were considered unrelated to treatment.

Dose-dependent hepatocellular periportal vacuolation in males and females correlated at 600 mg/kg bw/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5, respectively. Intracellular lipids accumulate when their rate of degradation no longer exceeds their rate of synthesis or their release into the bloodstream is slowed (see citations in the full study report). The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg bw/day. Examination of the liver in males and females, following two weeks without treatment revealed partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes. Therefore the changes were likely not to be adverse. In addition, a dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg bw/day. Hyaline droplets are composed of poorly catabolized α2μ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The higher incidence and multifocal nature of the basophilic tubules seen in this group most likely reflects damage and is adverse Examination of the kidneys from Control and males previously treated at 600 mg/kg bw/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Although basophilic tubules were seen in two Control males and one male receiving 150 mg/kg bw/day of the test item they were only present focally and were consistent with background levels.

Toxicity to humans is considered unlikely as little or no α2μ globulin is present in humans.

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:
At 300 and 600 mg/kg bw/day: T4 concentration in blood T4 concentration in blood from males treated for five weeks was statistically significantly lower than Control.
At 150 mg/kg bw/day: T4 concentration in blood from males was marginally lower than Control.
T4 concentrations in males treated at 600 mg/kg bw/day were comparable to Control and endogenous levels following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.
T4 concentrations in blood from male and female offspring were comparable with endogenous levels.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no effects on post implantation survival or litter size related to parental treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

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:

not specified

Description (incidence and severity):

Pre-coital interval, mating performance were unaffected by treatment. Litter size, was not affected by treatment.

Prior to treatment all females had regular estrus cycles of 4 to 5 days. All females had a regular estrus cycle of 4 to 5 days during the treatment period, with the exception of one at 600 mg/kg bw/day that had an irregular cycle, one at 600 mg/kg bw/day that had an extended estrus, and two at 300 mg/kg bw/day and one at 600 mg/kg bw/day that showed acyclicity. All non-mated females showed regular estrous cycles at the end of treatment and during the two week period of no treatment at 600 mg/kg bw/day.

Two females receiving 600 mg/kg bw/day that showed evidence of mating, failed to litter.

All females were in diestrus at termination on Day 13 of lactation.

Other effects:

not specified

Description (incidence and severity):

Two females receiving 600 mg/kg bw/day that showed evidence of mating, failed to litter.

At 600 mg/kg bw/day, two females were not pregnant and three litters died by Day 2 of age that was attributed to the reduced secretory activity of the mammary gland in F1 females. Two of these females exhibited irregularities of the estrous cycle before mating. Of the three females with litter loss, one showed extended estrus, and a second had an irregular cycle.

The offspring from adults treated at 600 mg/kg bw/day were small on Day 1 of age and subsequent growth, especially during Days 4-7, to Day 13 was poor and correlated with clinical signs of poor maternal care.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

changes in number of pregnant

Remarks on result:

other: precautionary NOAEL only. due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Which correlated with clinical signs of poor maternal care.

Abnormalities:

no effects observed

Description (incidence and severity):

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

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: body weight on Day 1 of age was markedly low, when compared with Control. Subsequent growth, especially during Days 4-7, was low and resulted in low overall (Days 1-13) body weight gains. On Day 13 of age, males were 14% smaller than Control animals.
At 300 mg/kg bw/day: body weight was unaffected by parental treatment
At 150 mg/kg bw/day: body weight was unaffected by parental treatment

Females:
At 600 mg/kg bw/day: body weight on Day 1 of age was markedly low, when compared with Control. Subsequent growth, especially during Days 4-7, was low and resulted in low overall (Days 1-13) body weight gains. On Day 13 of age, females were 17% smaller than Control animals.
At 300 mg/kg bw/day: body weight was unaffected by parental treatment
At 150 mg/kg bw/day: body weight was unaffected by parental treatment

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring survival

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring sex ratio

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring litter size.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

Two females receiving 600 mg/kg bw/day were not pregnant and three litters died by Day 2 of lactation; the maternal mammary glands of these females showed reduced secretory activity. Some offspring at 300 or 600 mg/kg bw/day had clinical signs that indicated poor maternal care.

External malformations:

no effects observed

Description (incidence and severity):

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

Ano-genital distances for male or female offspring were unaffected by treatment with test item. Male offspring did not develop nipples.

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

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in postnatal survival

Remarks on result:

other: precautionary NOAEL only. due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Which correlated with clinical signs of poor maternal care.

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 600 mg/kg bw/day. The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Applicant assessment indicates: that whilst the effects on lipid metabolism where not adverse in adults since they were deemed reversible, they may have led to adverse outcome in offspring via reduced secretory activity in female adults and consequential poor maternal care. On this basis a precautionary NOAEL has been adopted.

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), 150, 300 or 600 mg/kg bw/day test item formulated in corn oil vehicle. Ten males were treated in the 150 and 300 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 600 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 150, 300 or 600 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, ophthalmic changes and/or pre-coital interval, mating performance gestation length on the adult animals. Body weight gain was variable throughout the treatment period in males and non-mated females at 600 mg/kg bw/day. Overall gains were similar to control in both. Following cessation of treatment, females showed body weight loss, compared with body weight stasis in the Control. Body weight gain was variable during both gestation and lactation, but again overall gains in treated animals were similar to Control. Food consumption was similarly affected by treatment in all study phases, but was noticeably low during Days 4-10 of lactation. Water consumption was consistently high in males and non mated females at 300 or 600 mg/kg bw/day, during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day. There were no toxicologically significant changes in haematology, blood chemistry and plasma parameters following five weeks of treatment in males and unmated females and on Day 13 of lactation except for minor elevated plasma cholesterol concentration in non-mated females and plasma urea concentration in males and minor change in creatinine concentrations in males/females receiving 600 mg/kg bw/day. These changes were attributed to changes in microscopic changes observed in male kidney and/or male/female liver. At the end of treatment liver weight and kidney weight were high in males/females at 300 and 600 mg/kg bw/day and 600 mg/kg bw/day, respectively. Urinalysis indicated a high urinary volume as a result of the higher water intake in males/females at 600 mg/kg bw/day. This may be attributed to higher plasma and urinary protein concentrations. Macroscopic examination performed after 5 weeks of treatment or 2 weeks recovery or reproductive females after day 13 lactation revealed no test item treatment related lesions. Microscopic examination revealed a dose-dependent hepatocellular periportal vacuolation in males and females treated at 150, 300 or 600 mg/kg/day that correlated at 600 mg/kg/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5. This metabolic alteration may also be reflected by the presence of an increase in urinary ketones in all males at 600 mg/kg/day. The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg/day. Examination of the liver in males and females at 600 mg/kg/day, following two weeks without treatment indicated partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes, therefore changes were considered unlikely to be adverse. A dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg/day. Hyaline droplets are composed of poorly catabolized α2µ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The presence of increased α2µ globulin in males was confirmed immunohistochemically. Examination of the kidneys from Control and males previously treated at 600 mg/kg/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Basophilic tubules were only present focally and were consistent with background levels of this finding. Only partial recovery was evident following two weeks without treatment. Relevance for toxicity in humans through this α2µ globulin mechanism is considered unlikely.

 

At 600 mg/kg/day, two females were not pregnant. Three litters indicated mortality by Day 2, that was attributed to the reduced secretory activity of the mammary gland in the adults. Two females exhibited irregularities of the estrous cycle before mating. Of the three females with total litter loss, one showed extended estrus, and a second had an irregular cycle. The offspring from adults treated at 600 mg/kg/day were small on Day 1 of age and subsequent growth, especially during Days 4-7, to Day 13, correlated with clinical signs of poor maternal care. There was some evidence of poor maternal care at 300 mg/kg/day. Ano genital distance in the offspring was unaffected by maternal treatment. Male offspring did not develop nipples. T4 (thyroxine) levels were statistically low in males at 300 or 600 mg/kg bw/day but were comparable with control following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.

 

Conclusion:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 600 mg/kg bw/day. The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Applicant assessment indicates: that whilst the effects on lipid metabolism where not adverse in adults since they were deemed reversible, they may have led to adverse outcome in offspring via reduced secretory activity in female adults and consequential poor maternal care. On this basis a precautionary NOAEL has been adopted.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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: January 2018 ; signature: June 2018

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-methyl-4-propyl-1,3-oxathiane

EC Number:

261-699-8

EC Name:

cis-2-methyl-4-propyl-1,3-oxathiane

Cas Number:

59323-76-1

Molecular formula:

C8H16OS

IUPAC Name:

2-methyl-4-propyl-1,3-oxathiane

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: dry area protected from light, in a refrigerator (2 to 10 °C). Store under nitrogen in a closed container after first opening.
- 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), in the dark under nitrogen and under yellow light when handled.
(ii) Formulated: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre-vehicle formulation: Not applicable.
(ii) Formulated: In a preceding (1) formulation and method validation and (2) prior/within definitive testing: the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix. The stability was demonstrated for up to 15 days following refrigerated storage (2 to 8°C) and for 21 days following ambient storage (15 to 25°C). Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test item. In the dose formulation analysis, mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: The test item was found to be adequately soluble in corn oil vehicle formulations up to 200 mg/mL. The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Procedural recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. The mean concentrations of test item in formulations analysed for the study were within 10% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 1%, confirming precise analysis. Procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.
- 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. For each concentration, approximately 40% of the required volume of corn oil was added to the required amount of test item, and magnetically stirred until uniformly mixed. The remaining vehicle was added, and the formulation magnetically stirred until homogeneous.
- 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.

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

OTHER SPECIFICS: Not applicable.

Test animals

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 70 to 77 days old ; females 84 to 91 days old.
- Weight at study initiation: males 344 to 418 g ; females 242 to 308 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: seven days before commencement of treatment; females: 21 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: 2018-02-28 To: 2018-05-09

Administration / exposure

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 twenty-one (21) day at 15 to 25°C and up to fifteen (15) days at 2 to 8°C. Formulations were prepared weekly during the treatment period and stored at ambient (15 to 25°C), in amber glass bottles.

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: 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. Results show the formulations to be homogeneous and stable in vehicle.

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: Ambient (15 to 25°C), in amber glass bottles.
- Stability under test conditions:
(i) Pre- vehicle formulation: Stable ; formulated-test item in-vehicle was prepared weekly
(ii) Formulated-vehicle: up to fifteen (15) days at 2-8°C and was validated stable for twenty-one (21) days 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 twenty-one (21) days at ambient and up to fifteen (15) days refrigerated (2 to 8°C). Formulations were prepared weekly during the treatment period and stored at ambient (15 to 25°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% nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations of 2 mg/mL and 200 mg/mL, during distribution of bottles, magnetic stirring for 2 hours and ambient temperature storage for up to twenty-one (21) days and refrigerated storage for up to fifteen (15) 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% of nominal applied limits.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability: The formulation analysis, confirmed homogeneity and stability for the test item in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for up to 21 days and refrigerated storage (2 to 8ºC) for up to 15 days. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method, with the exception of one recovery prepared on Day 8 of the trial. This can be excluded as per laboratory SOP. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 21 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 10% of the initial time zero value and the coefficient of variation was less than 2%.
- The analysis consisted of GC-FID analysis with external calibration (within a dedicated formulation analysis report attached to the full study report). Samples in corn oil were accurately fortified with known amounts of test item in an end solution in teradecane. These were then subjected to analysis by GC-FID analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). With LOD = 0.0499 μg/mL; linearity = > 0.99 between 10 μg/mL and 100 μg/mL. Repeatability (n=6) of < 1%. Accuracy and precision was confirmed and mean procedural recovery was 97.6% (n=5 ; CV = 1.79%) at 2 mg/mL and 99.3% (CV = 0.89% ; n=5) at 200 mg/mL.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% 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 (females were not dosed if parturition was in progress, at the scheduled time of administration).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Doses / concentrations 4

Dose / conc.:

600 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
150 mg/kg bw/day (toxicity test) = 10
300 mg/kg bw/day (toxicity test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
150 mg/kg bw/day (reproduction test) = 10
150 mg/kg bw/day (toxicity test) = 5
300 mg/kg bw/day (reproduction test) = 10
300 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (reproduction test) = 10
600 mg/kg bw/day (toxicity test) = 5
600 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a previously conducted 14-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 14-day sighting test were: Group 1: control (vehicle: Corn oil), Group 2: 300 mg/kg bw/day, Group 3: 600 mg/kg bw/day and group 4: 1000 mg/kg bw/day. In the 14-day sighting test (administered consecutively, for 14-days) the following effects were determined. Group 4 (high dose: 1000 mg/kg bw/day) was terminated early due to marked toxicity (reduced body tone and uncoordinated gait). There were no clinical signs in the 300 or 600 mg/kg bw/day dose levels. At 600 mg/kg bw/day, males had slightly lower overall body weight gain. Males/females at 600 mg/kg bw/day and females only at 300 mg/kg bw/day had periods of slightly lower food intake but overall food consumption was unaffected. In both males and females, liver weights were higher with a dose relationship at 300 and 600 mg/kg bw/day. There were no adverse macroscopic findings. Basis: other: nominal in vehicle (corn oil)
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14-days
- Section schedule rationale: Random

Examinations

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study). Water consumption was recorded daily by weight for each cage of animals, using water bottles fitted with sipper tubes from the end of Week 2. Water consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males during Week 4. Water consumption was recorded continuously for Toxicity phase and Recovery phase females.
- 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.

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

Comparisons were performed:
Group 1 vs 2, 3 and 4

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).

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs observed that were considered adverse and related to treatment.

At 600 mg/kg bw/day: one male had piloerection on day 20, and transient salivation was evident following dosing in week 5 in four males. Transient salivation, with or without chin rubbing, was occasionally seen in females during gestation at 150, 300 and 600 mg/kg bw/day. There was no adverse effect on sensory reactivity and grip strength, motor activity.
For females in gestation and lactation:
Transient salivation, with or without chin rubbing, was occasionally seen during gestation at 150, 300 or 600 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities considered directly related to treatment.

One male treated at 300 mg/kg/day (M-18) was humanely terminated on day 22 for poor condition. Subject to necropsy. Microscopic examination revealed vacuolation of the periportal hepatocytes that was identified in animals terminated following five weeks of treatment. One female (F-58) experienced mortality during blood sampling on day 13. There were no relevant microscopic or macroscopic findings. This was not considered treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: body weight gain was variable throughout the treatment period in males and non-mated females. Overall gains were similar to control in both.
Females:
No significant findings. In non-mated males body weight gain was variable throughout the treatment period.
For females in gestation and lactation:
At 150, 300 or 600 mg/kg bw/day: body weight gain was variable but overall unaffected by treatment. During lactation, body weight gain was variable, low in days 4-7 and high in days 7-13. Overall bodyweight gain was comparable to Control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in males. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.
Females:
At 300 and 600 mg/kg bw/day: food consumption was variable throughout the treatment period in non-paired females. Low in week 1 compared to control and thereafter during treatment and recovery period: unaffected.
For females in gestation and lactation:
At 150, 300 and 600 mg/kg bw/day: food consumption was mostly similar to controls, with exception of 600 mg/kg bw/day where it was low during days 17 to 20. Food intact was low during day 4-10 lactation, but overall was considered unaffected.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Males:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in males. Over the full recovery period it was considered unaffected by treatment.
Females:
At 300 or 600 mg/kg bw/day: water consumption was consistently high in non mated females. Over the full recovery period it was considered low.
For females in gestation and lactation:
Water consumption was consistently high during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic changes.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in haematological parameters that would be considered adverse.

Examinations following two-week recovery and on day 13 of lactation did not identify any differences to control that were attributable to treatment. All differences to controls were considered minor or did not show dose-response relationships and therefore were considered the result of normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in blood chemistry parameters that would be considered adverse.

At 600 mg/kg bw/day: there was elevated plasma cholesterol concentration in non-mated females and elevated plasma urea concentration in males. It was considered these differences may be associated with test item related microscopic changes in the male kidney and the liver of both sexes at the corresponding dose level. Plasma urea concentration remained high in males following two-weeks without treatment.

See ‘Gross pathological findings’ and ‘histopathological findings – non-neoplastic’ for further information.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was higher but differences failed to show a dose response.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Females:
At 600 mg/kg bw/day: there was an increase in urinary ketones. Urinary volume was increased. Total urinary protein was higher. Creatine output was lower.
At 300 mg/kg bw/day: there was more than expected urinary ketones. Total urinary protein was higher.
Following two-week recovery and/or at Day 13 lactation: differences to controls were considered minor or the result of 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 or within historic control data ranges and/or were considered unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: liver weight and kidney weight were high
At 300 mg/kg bw/day: liver weight were high
Females:
At 600 mg/kg bw/day: liver weight and kidney weight were high
At 300 mg/kg bw/day: liver weight were high
At 150 mg/kg bw/day: liver weight were high
All other findings were considered minor or the result of normal biological variation.

Adjusted liver and kidney weights remained high, although less markedly, in males and females previously treated at 600 mg/kg/day when compared with Control. Absolute and adjusted organ weights were unaffected by treatment on Day 13 of lactation.

See ‘Gross pathological findings’ and ‘histopathological findings – non-neoplastic’ for further information.

Gross pathological findings:

not specified

Description (incidence and severity):

Macroscopic examination performed after 5 weeks of treatment or 2 weeks recovery or reproductive females after day 13 lactation revealed no test item treatment related lesions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all males ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of all males. Hyaline droplets were confirmed to be composed of α2μ globulin (immunohistochemistry). All males were graded as having minimal to slight α2μ globulin present.
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation ; Kidney: hyaline droplets and basophilic tubules were seen in the cortical tubules of three males
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
Females:
At 600 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation seen in all females
At 300 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation
At 150 mg/kg bw/day: Liver: Periportal hepatocyte vacuolation

All other findings were considered unrelated to treatment.

Dose-dependent hepatocellular periportal vacuolation in males and females correlated at 600 mg/kg bw/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5, respectively. Intracellular lipids accumulate when their rate of degradation no longer exceeds their rate of synthesis or their release into the bloodstream is slowed (see citations in the full study report). The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg bw/day. Examination of the liver in males and females, following two weeks without treatment revealed partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes. Therefore the changes were likely not to be adverse. In addition, a dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg bw/day. Hyaline droplets are composed of poorly catabolized α2μ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The higher incidence and multifocal nature of the basophilic tubules seen in this group most likely reflects damage and is adverse Examination of the kidneys from Control and males previously treated at 600 mg/kg bw/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Although basophilic tubules were seen in two Control males and one male receiving 150 mg/kg bw/day of the test item they were only present focally and were consistent with background levels.

Toxicity to humans is considered unlikely as little or no α2μ globulin is present in humans.

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:
At 300 and 600 mg/kg bw/day: T4 concentration in blood T4 concentration in blood from males treated for five weeks was statistically significantly lower than Control.
At 150 mg/kg bw/day: T4 concentration in blood from males was marginally lower than Control.
T4 concentrations in males treated at 600 mg/kg/day were comparable to Control and endogenous levels following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.
T4 concentrations in blood from male and female offspring were comparable with endogenous levels.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 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

Remarks:

The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely.

Target system / organ toxicity

Critical 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 600 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), 150, 300 or 600 mg/kg bw/day test item formulated in corn oil vehicle. Ten males were treated in the 150 and 300 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 600 mg/kg bw/day. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 150, 300 or 600 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, ophthalmic changes and/or pre-coital interval, mating performance gestation length on the adult animals. Body weight gain was variable throughout the treatment period in males and non-mated females at 600 mg/kg bw/day. Overall gains were similar to control in both. Following cessation of treatment, females showed body weight loss, compared with body weight stasis in the Control. Body weight gain was variable during both gestation and lactation, but again overall gains in treated animals were similar to Control. Food consumption was similarly affected by treatment in all study phases, but was noticeably low during Days 4-10 of lactation. Water consumption was consistently high in males and non mated females at 300 or 600 mg/kg bw/day, during gestation at 600 mg/kg bw/day and lactation at 300 or 600 mg/kg bw/day. There were no toxicologically significant changes in haematology, blood chemistry and plasma parameters following five weeks of treatment in males and unmated females and on Day 13 of lactation except for minor elevated plasma cholesterol concentration in non-mated females and plasma urea concentration in males and minor change in creatinine concentrations in males/females receiving 600 mg/kg bw/day. These changes were attributed to changes in microscopic changes observed in male kidney and/or male/female liver. At the end of treatment liver weight and kidney weight were high in males/females at 300 and 600 mg/kg bw/day and 600 mg/kg bw/day, respectively. Urinalysis indicated a high urinary volume as a result of the higher water intake in males/females at 600 mg/kg bw/day. This may be attributed to higher plasma and urinary protein concentrations. Macroscopic examination performed after 5 weeks of treatment or 2 weeks recovery or reproductive females after day 13 lactation revealed no test item treatment related lesions. Microscopic examination revealed a dose-dependent hepatocellular periportal vacuolation in males and females treated at 150, 300 or 600 mg/kg/day that correlated at 600 mg/kg/day with increased storage of lipids within the cytoplasm of the hepatocytes and minor elevation of plasma cholesterol and elevated urea concentration in females or males in Week 5. This metabolic alteration may also be reflected by the presence of an increase in urinary ketones in all males at 600 mg/kg/day. The presence of periportal vacuolation correlated with the statistically significant increase in liver weights in males and females at 600 mg/kg/day. Examination of the liver in males and females at 600 mg/kg/day, following two weeks without treatment indicated partial recovery of the hepatocellular periportal vacuolation and normalisation of plasma cholesterol and urea concentrations. This treatment related change was not accompanied by inflammation or necrosis of the hepatocytes, therefore changes were considered unlikely to be adverse. A dose-dependent accumulation of hyaline droplets and basophilic tubules was evident in males receiving 300 or 600 mg/kg/day. Hyaline droplets are composed of poorly catabolized α2µ globulin probably binding to the test item and accumulating within the phagolysosomes of the renal tubular cells. The presence of increased α2µ globulin in males was confirmed immunohistochemically. Examination of the kidneys from Control and males previously treated at 600 mg/kg/day revealed resolution of the hyaline droplets however basophilic tubules persisted in the previously treated males. Basophilic tubules were only present focally and were consistent with background levels of this finding. Only partial recovery was evident following two weeks without treatment. Relevance for toxicity in humans through this α2µ globulin mechanism is considered unlikely.

 

At 600 mg/kg/day, two females were not pregnant. Three litters indicated mortality by Day 2, that was attributed to the reduced secretory activity of the mammary gland in the adults. Two females exhibited irregularities of the estrous cycle before mating. Of the three females with total litter loss, one showed extended estrus, and a second had an irregular cycle. The offspring from adults treated at 600 mg/kg/day were small on Day 1 of age and subsequent growth, especially during Days 4-7, to Day 13, correlated with clinical signs of poor maternal care. There was some evidence of poor maternal care at 300 mg/kg/day. Ano genital distance in the offspring was unaffected by maternal treatment. Male offspring did not develop nipples. T4 (thyroxine) levels were statistically low in males at 300 or 600 mg/kg bw/day but were comparable with control following two weeks without treatment. This change was reversible and with no pathological correlations was considered non-adverse.

 

Conclusion:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 600 mg/kg bw/day. The α2µ globulin mediated kidney changes were deemed adverse, in the rat. However, toxicity to humans by this mechanism is considered unlikely. For reproductive / developmental toxicity for males and females a precautionary NOAEL is set at 300 mg/kg bw/day due to low pregnancy rate and high litter loss reported at 600 mg/kg bw/day. Applicant assessment indicates: that whilst the effects on lipid metabolism where not adverse in adults since they were deemed reversible, they may have led to adverse outcome in offspring via reduced secretory activity in female adults and consequential poor maternal care. On this basis a precautionary NOAEL has been adopted.