Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized

Developmental toxicity / teratogenicity

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-diet formulation: Refrigerated (2 to 8°C), in the dark under nitrogen
(ii) Formulated-diet: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

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

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

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

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

other: 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 337 to 400 g ; females 243 to 315 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: 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: 2017-11-08 To: 2018-01-04

Route of administration:

oral: feed

Details on route of administration:

The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item and corn oil required for the premix were added to an equal amount of plain (basal) diet and stirred. An amount of plain diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a Turbula mixer for 100 cycles.
This premix was diluted with further quantities of plain diet using the doubling up process to prepare the required concentration test mixes. Each formulation was mixed using a Turbula mixer for 100 cycles.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified) with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1.
- Storage temperature of food: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 100 ppm and 20000 ppm versus an internal standard: Tetradecane at ca. 20 µg/mL added to each 1000 µL sample) within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 0.883 μg/mL and LOQ = 2.942 μg/mL; linearity = > 0.998 between 5 μg/mL and 50 μg/mL. Repeatability (n=6) of < 3%. Accuracy and precision was confirmed and mean procedural recovery was 106.9 % (n=5 ; CV = 0.99%) at 100 ppm and 104.2% (CV = 0.47; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet with corn oil stabilizer formulations at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 28 hours and ambient/frozen temperature (-10 to -30°C) for up to 22 days (1 day ambient then 21 days frozen) following fresh preparation.
- Mean concentrations of diet-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/diet formulation. Specifically, samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item. The mean concentrations of test item were within 8% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 5%, confirming precise analysis.

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:

Continuous via diet

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet + corn oil

Dose / conc.:

1 600 ppm

Remarks:

Low – Group 2

Dose / conc.:

5 200 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

12 000 ppm

Remarks:

High – Group 4

Dose / conc.:

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

Remarks:

Mean for males for 1600 ppm group

Dose / conc.:

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

Remarks:

Mean for males for 5200 ppm group

Dose / conc.:

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

Remarks:

Mean for males for 12000 ppm group

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 1600 ppm

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 5200 ppm

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 12000 ppm group

No. of animals per sex per dose:

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
1600 ppm (toxicity test) = 10
5200 ppm (toxicity test) = 10
12000 ppm (toxicity test) = 5
12000 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
1600 ppm (reproduction test) = 10
1600 ppm (toxicity test) = 5
5200 ppm (reproduction test) = 10
5200 ppm (toxicity test) = 5
12000 ppm (reproduction test) = 10
12000 ppm (toxicity test) = 5
12000 ppm (recovery period) = 5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1600, 5200 and 12000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ±20% of the mean for the appropriate sex and/or due to poor condition or ill health.
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random

Observations and examinations performed and frequency:

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

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Mean daily consumption per animal (g/animal/day) was calculated for each phase
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Achieved dose was calculated using the mid-period body weight.
- Other: Food consumption was recorded for F0: Daily (including recovery phase). Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 19) but recommenced for males on Day 20. Reproductive females after mating food consumption was recorded daily until Day 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)
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No, although eyes were subject to pathology procedures at necropsy
- Time schedule for examinations: At termination only.
- Dose groups that were examined: All groups.

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

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

URINALYSIS: No.

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

IMMUNOLOGY: No

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

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

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

Sacrifice and pathology:

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

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

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

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

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

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

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

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

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

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

There were no treatment related mortalities.
There were three mortalities in the Control females of the reproductive phase. It was considered, the major factor contributing to death in all decedents was difficulties arising from parturition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, overall body weight gain during the treatment period (Day 1 to 43) was significantly low (82% of control).
At the beginning of the recovery period the bodyweights for males previously treated at 12000 ppm were slightly lower than control (96%), however by Day 15 of recovery they had returned to similar levels (99% of control). There was evidence of recovery.
Females:
There was no effect on overall body weight gain (Day 1 to 43) in females of the toxicity phase.

Recovery phase females, receiving 12000 ppm during the treatment period, had similar body weights to Control at the start of the recovery phase; by the end of the recovery period, the treated females had markedly higher body weight gains (four-fold increase to Control).

At 12000 ppm, there was a mean weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15.
At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control.
At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental.

For females in gestation:
At 12000 ppm had significantly low body weight at the start of gestation, mean gain over Days 0-7 was similar to Control. Their overall body weight gain (gestation day 0 to 20) was low (86%), when compared to control. Only the body weight change between Day 7 to 14 (82%) for females receiving 12000 ppm achieved statistical significance, however the change between Day 14 to 20 was also low (81%). Females at 1600 or 5200 ppm were unaffected during the gestation period.
For females in lactation:
Receiving 12000 ppm or 5200 ppm: overall body weight gain (1 to 13 of lactation) was low in females receiving, and this was largely influenced by the low body weight gain from Day 1 to 4 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Achieved doses generally maintained the intervals between dietary concentrations and increased during lactations due to increased physiological demand.
Males:
At 12000 ppm, food consumption was consistently lower than controls during the treatment period, particularly on Days 1 to 3 of treatment, although the extent of the difference diminished with time. During the recovery period, food consumption for males previously receiving 12000 ppm was slightly higher than control.
At 5200 ppm, males had lower intake on Day 1 with no consistent differences thereafter.
At 1600 ppm, no food consumption affects were observed.
Females:
At 12000 ppm, lower food intake in the first week of treatment achieving statistical significance at p < 0.01 level. During the recovery period, food consumption for females previously receiving 12000 ppm was generally slightly lower than in control.
At 5200 ppm, lower food intake was observed particularly on the first 2 days of treatment.
At 1600 ppm, lower food intake was observed particularly on the first 2 days of treatment.

For females in gestation:
Receiving 12000 ppm or 5200 ppm or 1600 ppm: food consumption tended to be marginally higher than control with once incidence of statistical significance.
For females in lactation:
Receiving 12000 ppm: food consumption was low from lactation Day 3 onwards
Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control. There was evidence of recovery.
Females:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control.

However there were no significant differences from control of erythrocyte count in males, or of hematocrit and hemoglobin concentration in both male and female recovery animals. There was evidence of recovery.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, low aspartate aminotransferase, high glucose and low potassium concentration and low bile acids, when compared with control.
Low aspartate aminotransferase was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.
Females:
At 12000 ppm, low alkaline phosphatase or phosphorus concentrations and low bile acids and low glucose, when compared with control.
Low alkaline phosphatase or phosphorus was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.

Urinalysis findings:

not examined

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, decreased terminal body weights were evident in males. Analysis of organ weights for the Toxicity phase animals revealed increased absolute and adjusted liver weights in all treated groups, with only males receiving 1600 ppm not achieving statistical significance. Recovery phase males previously treated at 12000 ppm had higher than control adjusted kidney weights, although this was considered as being reflected from a slightly lower than normal control value.
Females:
At 12000 ppm, Absolute and adjusted weights for the uterus, cervix and oviducts were low in all groups of treated toxicity females. This was not evident in reproductive phase females. It was considered as being reflected from a high control value. The difference in uterus, cervix and oviducts was not apparent in the reproductive phase females.
Absolute and adjusted ovary weights were low in reproductive phase females treated at 12000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no significant pathological findings observed that were considered related to treatment or were observed in the recovery phase groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, Liver: Centrilobular hypertrophy. Thyroid: Follicular cell hypertrophy. Kidney: Accumulation of hyaline droplets
Females:
At 12000 ppm, Liver: Centrilobular hypertrophy.
There were no findings that were considered to be treatment related after 2 weeks of recovery.

Accumulation of hyaline droplets was seen in males that received a dietary concentration of 12000 ppm. Positive diffuse immunostaining for alpha-2u globulin was seen in all control males and males that received a dietary concentration of 12000 ppm at normal background levels. However, positive multifocal immunostaining for alpha-2u globulin was only seen in males that received a dietary concentration of 12000 ppm indicating increased accumulation of alpha-2u globulin compared to control animals. Toxicity in humans through this mechanism is considered improbable as little or no α2u globulin is present in humans, and therefore these findings in the male rat kidney were considered to be of no relevance to man. Variations in plasma glucose, potassium and phosphorus were reported in the high dietary concentration although there was no significant increase in kidney weights at 12000 ppm.

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:
Males:
At 12000 ppm, serum T4 levels were low in F0 males and slightly low in offspring. There was evidence of recovery.
At 5200 ppm, serum T4 levels were slightly low in F0 males. There was evidence of recovery.
During recovery, T4 concentrations in adult males returned to control levels.

There was considered to be no clear decrease seen T4 levels of offspring - review of the individual values did not reveal a convincing effect of treatment with mean hormone levels at 12000 ppm within the expected range.

There were no clear differences in Serum T3 levels in either the adult males or offspring on Day 13 of age; any differences from Controls were relatively minor, lacked relationship to dose and, therefore, attributed to normal biological variation.

No further analysis of T3/T4 or TSH was considered necessary.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 12 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 663 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 668 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 12000 ppm which was equivalent to actual dose received: 663 mg/kg body weight per day (males) and 668 mg/kg body weight per day (females).

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least six weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received dietary test item at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. Ten males were treated in the 1600 and 5200 ppm doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 12000 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. 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 13 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Mean achieved doses for males at 1600, 5200 or 12000 ppm were 93.7, 303 or 663 mg/kg/day, respectively. For females of the toxicity and recovery phase.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no effect on clinical condition, sensory reactivity and grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. At 12000 ppm, there was a mean body weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15. At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control. At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental. Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size. For females at 12000 ppm, body weight gains during gestation or lactation were slightly lower than expected given the litter size and comparison between groups. Over the duration of the study, overall mean body weight gain was only lower in males at 12000 ppm. This coincides with the pattern of food intake seen in the earlier and later part of the study. Hematological investigations at the end of the treatment period revealed lower hematocrit, hemoglobin concentration and erythrocyte count in animals at 12000 ppm. There were no differences in these parameters following a 14 day recovery period. Blood chemistry investigations at the end of the treatment period revealed, in males, low aspartate aminotransferase in all treated groups, high glucose and potassium concentration at 12000 ppm and low bile acids at 12000 ppm. And for females, low alkaline phosphatase or phosphorus concentrations in all treated groups and low bile acids and glucose concentrations at 12000 ppm. There were no differences in these parameters following the 14 day recovery period.

 

At scheduled termination of the Toxicity phase animals after 6 weeks of treatment, decreased terminal body weights were evident in males treated at 12000 ppm. Analysis of organ weights for the Toxicity phase animals revealed high liver weights in all treated groups. After 14 days of recovery, Recovery phase males previously treated at 12000 ppm had high kidney weights. Microscopic changes related to treatment with the test item were seen in the liver of both sexes and the thyroid and kidneys of males. Centrilobular hepatocyte hypertrophy was seen in animals that received a dietary concentration of 12000 ppm. Follicular cell hypertrophy was seen in the thyroids of males that received a dietary concentration of 12000 ppm. Accumulation of hyaline droplets was seen in the kidneys of males that received a dietary concentration of 12000 ppm. There were no findings that were considered to be treatment related after 2 weeks of recovery. Levels of circulating thyroxine obtained from Main study male animals were low at 12000 ppm and further assessment of Recovery study males animals after a 2 week recovery period revealed no differences from Control after 2 weeks of no treatment. There were no differences from Control for levels of circulating Triiodothyronine (T3) in adult males or from F1 offspring on Day 13 of age.

 

Conclusion:

Dietary administration of test item to CD rats at concentrations up to and including 12000 ppm was generally well tolerated. Test item related histopathological changes were apparent in the kidney and thyroid of males and the liver of both sexes given 12000 ppm. The kidney changes detected in the males (hyaline droplets in tubules) were consistent with species-specific responses of the male rat in response to xenobiotics, and therefore may be of little value as an indicator of hazard to human health. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) in both sexes and associated thyroid changes (follicular cell hypertrophy) and lower T4 levels in males are deemed reversible. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was concluded to be 12000 ppm (equivalent to 663 mg/kg/day for males and 668 mg/kg/day for females).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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: December 2016 ; signature: March 2017

Limit test:

no

Specific details on test material used for the study:

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) Pre-diet formulation: Refrigerated (2 to 8°C), in the dark under nitrogen
(ii) Formulated-diet: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

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

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

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

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

other: 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 337 to 400 g ; females 243 to 315 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: 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: 2017-11-08 To: 2018-01-04

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified) with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1.
- Storage temperature of food: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 100 ppm and 20000 ppm versus an internal standard: Tetradecane at ca. 20 µg/mL added to each 1000 µL sample) within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 0.883 μg/mL and LOQ = 2.942 μg/mL; linearity = > 0.998 between 5 μg/mL and 50 μg/mL. Repeatability (n=6) of < 3%. Accuracy and precision was confirmed and mean procedural recovery was 106.9 % (n=5 ; CV = 0.99%) at 100 ppm and 104.2% (CV = 0.47; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet with corn oil stabilizer formulations at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 28 hours and ambient/frozen temperature (-10 to -30°C) for up to 22 days (1 day ambient then 21 days frozen) following fresh preparation.
- Mean concentrations of diet-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/diet formulation. Specifically, samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item. The mean concentrations of test item were within 8% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 5%, confirming precise analysis.

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 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 ppm

Remarks:

Control – Group 1; Basal diet + corn oil

Dose / conc.:

1 600 ppm

Remarks:

Low – Group 2

Dose / conc.:

5 200 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

12 000 ppm

Remarks:

High – Group 4

Dose / conc.:

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

Remarks:

Mean for males for 1600 ppm group

Dose / conc.:

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

Remarks:

Mean for males for 5200 ppm group

Dose / conc.:

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

Remarks:

Mean for males for 12000 ppm group

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 1600 ppm

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 5200 ppm

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 12000 ppm group

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (before pairing) for 1600 ppm

Dose / conc.:

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

Remarks:

500 - 700 mg/kg bw/day (actual dose received)
Mean for reproductive phase females (before pairing) for 5200 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (before pairing) for 12000 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 1600 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 5200 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 12000 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (lactation) for 1600 ppm

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (lactation) for 5200 ppm

Dose / conc.:

1 627 mg/kg bw/day (actual dose received)

Remarks:

Mean for reproductive phase females (lactation) for 12000 ppm

No. of animals per sex per dose:

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

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1600, 5200 and 12000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ±20% of the mean for the appropriate sex and/or due to poor condition or ill health.
Other:
- 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: Yes. Mean daily consumption per animal (g/animal/day) was calculated for each phase
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Achieved dose was calculated using the mid-period body weight.
- Other: Food consumption was recorded for F0: Daily (including recovery phase). Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 19) but recommenced for males on Day 20. Reproductive females after mating food consumption was recorded daily until Day 12 of lactation.

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

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. THYROID HORMONE ANALYSIS: Yes
- Time schedule:
(i) at day 4 of age, F1 offspring, two females per litter (where possible) - no females were selected when total litter size dropped below ten/litter or if the resultant number of live females were to be less than 3 for subsequent day 13 procedures. One F1 female for T3/T4 (serum) and One F1 female for TSH (plasma).
(ii) At day 13 of age, F1 offspring, two males and two females per litter (where possible) – one male and one female for T3/T4 (serum) where possible and one male and one female for TSH (plasma) where possible
(iii) at 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 beginning of treatment until pairing
- Wet smears – All females (including spares): taken for 14 days before treatment; females that failed to exhibit 4-5 day cycles were not allocated to the study ; Reproductive females only: after pairing until mating ; All females: four days before scheduled termination

Sperm parameters (parental animals):

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

Litter observations:

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

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

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 Males: After 5 weeks treatment.
- Maternal animals: F0 females failing to produce a viable litter: Day 25 after mating. F0 females: Day 14 of lactation.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues were prepared for microscopic examination and weighed, respectively. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed at scheduled intervals.
- Immunohistochemistry - Kidneys : All Toxicity phase male animals from Group 1 and 4 were assessed for immunostaining for for alpha-2u globulin.

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.

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).

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

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

Reproductive indices:

Mating performance, fertility, gestation length, gestation index

Offspring viability indices:

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

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, overall body weight gain during the treatment period (Day 1 to 43) was significantly low (82% of control).
At the beginning of the recovery period the bodyweights for males previously treated at 12000 ppm were slightly lower than control (96%), however by Day 15 of recovery they had returned to similar levels (99% of control). There was evidence of recovery.
Females:
There was no effect on overall body weight gain (Day 1 to 43) in females of the toxicity phase.

Recovery phase females, receiving 12000 ppm during the treatment period, had similar body weights to Control at the start of the recovery phase; by the end of the recovery period, the treated females had markedly higher body weight gains (four-fold increase to Control).

At 12000 ppm, there was a mean weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15.
At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control.
At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental.

For females in gestation:
Receiving 12000 ppm had significantly low body weight at the start of gestation, mean gain over Days 0-7 was similar to Control. Their overall body weight gain (gestation day 0 to 20) was low (86%), when compared to control. Only the body weight change between Day 7 to 14 (82%) for females receiving 12000 ppm achieved statistical significance, however the change between Day 14 to 20 was also low (81%). Females at 1600 or 5200 ppm were unaffected during the gestation period.
For females in lactation:
Receiving 12000 ppm or 5200 ppm: overall body weight gain (1 to 13 of lactation) was low in females receiving, and this was largely influenced by the low body weight gain from Day 1 to 4 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Achieved doses generally maintained the intervals between dietary concentrations and increased during lactations due to increased physiological demand.
Males:
At 12000 ppm, food consumption was consistently lower than controls during the treatment period, particularly on Days 1 to 3 of treatment, although the extent of the difference diminished with time. During the recovery period, food consumption for males previously receiving 12000 ppm was slightly higher than control.
At 5200 ppm, males had lower intake on Day 1 with no consistent differences thereafter.
At 1600 ppm, no food consumption affects were observed.
Females:
At 12000 ppm, lower food intake in the first week of treatment achieving statistical significance at p < 0.01 level. During the recovery period, food consumption for females previously receiving 12000 ppm was generally slightly lower than in control.
At 5200 ppm, lower food intake was observed particularly on the first 2 days of treatment.
At 1600 ppm, lower food intake was observed particularly on the first 2 days of treatment.

For females in gestation:
Receiving 12000 ppm or 5200 ppm or 1600 ppm: food consumption tended to be marginally higher than control with once incidence of statistical significance.
For females in lactation:
Receiving 12000 ppm: food consumption was low from lactation Day 3 onwards
Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control. There was evidence of recovery.
Females:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control.

However there were no significant differences from control of erythrocyte count in males, or of hematocrit and hemoglobin concentration in both male and female recovery animals. There was evidence of recovery.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, low aspartate aminotransferase, high glucose and low potassium concentration and low bile acids, when compared with control.
Low aspartate aminotransferase was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.
Females:
At 12000 ppm, low alkaline phosphatase or phosphorus concentrations and low bile acids and low glucose, when compared with control.
Low alkaline phosphatase or phosphorus was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.

Urinalysis findings:

not examined

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.

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 12000 ppm, Liver: Centrilobular hypertrophy. Thyroid: Follicular cell hypertrophy. Kidney: Accumulation of hyaline droplets
Females:
At 12000 ppm, Liver: Centrilobular hypertrophy.
There were no findings that were considered to be treatment related after 2 weeks of recovery.

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:
Males:
At 12000 ppm, serum T4 levels were low in F0 males and slightly low in offspring. There was evidence of recovery.
At 5200 ppm, serum T4 levels were slightly low in F0 males. There was evidence of recovery.
During recovery, T4 concentrations in adult males returned to control levels.

There was considered to be no clear decrease seen T4 levels of offspring - review of the individual values did not reveal a convincing effect of treatment with mean hormone levels at 12000 ppm within the expected range.

There were no clear differences in Serum T3 levels in either the adult males or offspring on Day 13 of age; any differences from Controls were relatively minor, lacked relationship to dose and, therefore, attributed to normal biological variation.

No further analysis of T3/T4 or TSH was considered necessary.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Prior to the start of treatment all females assigned to all study phases showed regular 4-5 day estrous cycles.
There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females. All females showed regular 4-5 day cycles with exception to one control female and on female in the 1600 ppm group with irregular cycles; these irregular cycles had no impact upon pre-coital interval.

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

There were slightly low implantation counts in the females receiving 12000 ppm, compared to control and the historical control data range. It was considered that this reflected only one animal (An 0150) with only 4 implants.
By oversight, corpora lutea were not counted. However, in the absence of an effect of the number of implantations, this oversight had no effect on the study validity.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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

Reproductive performance:

no effects observed

Description (incidence and severity):

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 12 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 663 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Effect level:

>= 668 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

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

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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:

no effects observed

Description (incidence and severity):

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

Ano-genital distances for male or female offspring were unaffected by treatment with test item. There was higher incidence of litters/offspring containing males with nipples in the 12000 ppm group, when compared to control. The values were within the historical control data range.

Histopathological findings:

no effects observed

Description (incidence and severity):

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

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

Other effects:

no effects observed

Description (incidence and severity):

1. THYROID HORMONE ANALYSIS
F0 Males:
At 12000 ppm, serum T4 levels were low in F0 males and slightly low in offspring. There was evidence of recovery.
At 5200 ppm, serum T4 levels were slightly low in F0 males. There was evidence of recovery.
F1 offspring:
Analyses of samples for thyroxine (T4) obtained from F1 offspring on Day 13 of age were slightly low at 12000 or 5200 ppm

There were no clear differences in Serum T3 levels in either the adult males or offspring on Day 13 of age; any differences from Controls were relatively minor, lacked relationship to dose and, therefore, attributed to normal biological variation. No further analysis of T3/T4 or TSH was considered necessary.

In the related histopathology:

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

2. Offspring Ano-Genital Distance and Male Nipple Counts
Ano-genital distances for male or female offspring were unaffected by treatment with test item. There was higher incidence of litters/offspring containing males with nipples in the 12000 ppm group, when compared to control. The values were within the historical control data range.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 12 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Remarks:

gestation

Generation:

F1

Effect level:

>= 760 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Key result

Dose descriptor:

NOAEL

Remarks:

lactation

Generation:

F1

Effect level:

>= 1 627 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 12000 ppm, which was equivalent to actual dose received: 760 mg/kg body weight per day (gestation) and 1627 mg/kg body weight per day (lactation).

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least six weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received dietary test item at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. Ten males were treated in the 1600 and 5200 ppm doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 12000 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. 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 13 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. 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, untreated (no test item) diet, throughout the same relative treatment period. Mean achieved doses for males at 1600, 5200 or 12000 ppm were 93.7, 303 or 663 mg/kg/day, respectively. For females of the toxicity and recovery phase and reproductive phase females before pairing, mean achieved doses were 94.4, 296 or 668 mg/kg/day, respectively and ca. 100, 300 or 500-700 mg/kg/day for females before pairing. For females, mean achieved doses were 104, 322 and 760 mg/kg/day during gestation and 234.6, 747 or 1627 mg/kg/day during lactation at 1600, 5200 or 12000 ppm, respectively.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no effect on clinical condition, sensory reactivity and grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals.At 12000 ppm, there was a mean body weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15. At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control. At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental. Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size. For females at 12000 ppm, body weight gains during gestation or lactation were slightly lower than expected given the litter size and comparison between groups. Over the duration of the study, overall mean body weight gain was only lower in males at 12000 ppm. This coincides with the pattern of food intake seen in the earlier and later part of the study. Hematological investigations at the end of the treatment period revealed lower hematocrit, hemoglobin concentration and erythrocyte count in animals at 12000 ppm. There were no differences in these parameters following a 14 day recovery period. Blood chemistry investigations at the end of the treatment period revealed, in males, low aspartate aminotransferase in all treated groups, high glucose and potassium concentration at 12000 ppm and low bile acids at 12000 ppm. And for females, low alkaline phosphatase or phosphorus concentrations in all treated groups and low bile acids and glucose concentrations at 12000 ppm. There were no differences in these parameters following the 14 day recovery period.

 

At scheduled termination of the Toxicity phase animals after 6 weeks of treatment, decreased terminal body weights were evident in males treated at 12000 ppm. Analysis of organ weights for the Toxicity phase animals revealed high liver weights in all treated groups. After 14 days of recovery, Recovery phase males previously treated at 12000 ppm had high kidney weights. Microscopic changes related to treatment with the test item were seen in the liver of both sexes and the thyroid and kidneys of males. Centrilobular hepatocyte hypertrophy was seen in animals that received a dietary concentration of 12000 ppm. Follicular cell hypertrophy was seen in the thyroids of males that received a dietary concentration of 12000 ppm. Accumulation of hyaline droplets was seen in the kidneys of males that received a dietary concentration of 12000 ppm. There were no findings that were considered to be treatment related after 2 weeks of recovery. Levels of circulating thyroxine obtained from Main study male animals were low at 12000 ppm and further assessment of Recovery study males animals after a 2 week recovery period revealed no differences from Control after 2 weeks of no treatment. Analyses of samples for thyroxine (T4) obtained from F1 offspring on Day 13 of age were slightly low at 12000 or 5200 ppm. There were no differences from Control for levels of circulating Triiodothyronine (T3) in adult males or from F1 offspring on Day 13 of age. There was no effect of parental treatment on litter size, offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, nipple counts or macropathology. Macroscopic examination of offspring that died prior to scheduled termination or were killed at Day 13 of age did not reveal any findings that could be related to parental treatment.

 

Conclusion:

Dietary administration of test item to CD rats at concentrations up to and including 12000 ppm was generally well tolerated. Test item related histopathological changes were apparent in the kidney and thyroid of males and the liver of both sexes given 12000 ppm. The kidney changes detected in the males (hyaline droplets in tubules) were consistent with species-specific responses of the male rat in response to xenobiotics, and therefore may be of little value as an indicator of hazard to human health. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) in both sexes and associated thyroid changes (follicular cell hypertrophy) and lower T4 levels in males are deemed reversible. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was concluded to be 12000 ppm (equivalent to 663 mg/kg/day for males and 668 mg/kg/day for females), and the No Observed Adverse Effect Level (NOAEL) for the reproductive/developmental toxicity was concluded to be 12000 ppm (equivalent to 760 mg/kg/day during gestation and 1627 mg/kg/day during lactation).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: December 2016 ; signature: March 2017

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized

EC Number:

942-422-6

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Reaction products of ((5E)-5-ethylidenebicyclo[2.2.1]hept-2-ene and (5Z)-5-ethylidenebicyclo[2.2.1]hept-2-ene) and 2-methyl-1,3-butadiene, epoxidized

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Refrigerated (2 to 8°C), in the dark under nitrogen.
- Other: Colourless

Specific details on test material used for the study:

- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males 70 to 77 days old ; females 84 to 91 days old.
- Weight at study initiation: males 337 to 400 g ; females 243 to 315 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: 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: 2017-11-08 To: 2018-01-04

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified) with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1.
- Storage temperature of food: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable (for up to 22 days when stored frozen); formulated-diet was prepared weekly
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test, the test item formulated-diet was adequate stability and homogeneity for 22 days when formulations were stored frozen (-10 to -30°C) and 28 hours when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure. The same procedure was applied in the definitive test (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet of: Basal diet (SDS VRF1 Certified) with corn oil stabilizer at a ratio of 5 to 1 of test item to Corn Oil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 100 ppm and 20000 ppm versus an internal standard: Tetradecane at ca. 20 µg/mL added to each 1000 µL sample) within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With LOD = 0.883 μg/mL and LOQ = 2.942 μg/mL; linearity = > 0.998 between 5 μg/mL and 50 μg/mL. Repeatability (n=6) of < 3%. Accuracy and precision was confirmed and mean procedural recovery was 106.9 % (n=5 ; CV = 0.99%) at 100 ppm and 104.2% (CV = 0.47; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet with corn oil stabilizer formulations at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 28 hours and ambient/frozen temperature (-10 to -30°C) for up to 22 days (1 day ambient then 21 days frozen) following fresh preparation.
- Mean concentrations of diet-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/diet formulation. Specifically, samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item. The mean concentrations of test item were within 8% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 5%, confirming precise analysis.

Details on mating procedure:

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

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

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

Duration of test:

- Age at mating of the mated animals in the study: F0 generation: Males: ca. 12 weeks ; Females: ca. 13 weeks. (i.e. after two weeks treatment).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet + corn oil

Doses / concentrations 2

Dose / conc.:

1 600 ppm

Remarks:

Low – Group 2

Doses / concentrations 3

Dose / conc.:

5 200 ppm

Remarks:

Intermediate – Group 3

Doses / concentrations 4

Dose / conc.:

12 000 ppm

Remarks:

High – Group 4

Doses / concentrations 5

Dose / conc.:

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

Remarks:

Mean for males for 1600 ppm group

Doses / concentrations 6

Dose / conc.:

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

Remarks:

Mean for males for 5200 ppm group

Doses / concentrations 7

Dose / conc.:

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

Remarks:

Mean for males for 12000 ppm group

Doses / concentrations 8

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 1600 ppm

Doses / concentrations 9

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 5200 ppm

Doses / concentrations 10

Dose / conc.:

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

Remarks:

Mean for females (before pairing) for 12000 ppm group

Doses / concentrations 11

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (before pairing) for 1600 ppm

Doses / concentrations 12

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (before pairing) for 5200 ppm

Doses / concentrations 13

Dose / conc.:

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

Remarks:

500 - 700 mg/kg bw/day (actual dose received)
Mean for reproductive phase females (before pairing) for 12000 ppm

Doses / concentrations 14

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 1600 ppm

Doses / concentrations 15

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 5200 ppm

Doses / concentrations 16

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (gestation) for 12000 ppm

Doses / concentrations 17

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (lactation) for 1600 ppm

Doses / concentrations 18

Dose / conc.:

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

Remarks:

Mean for reproductive phase females (lactation) for 5200 ppm

Doses / concentrations 19

Dose / conc.:

1 627 mg/kg bw/day (actual dose received)

Remarks:

Mean for reproductive phase females (lactation) for 12000 ppm

No. of animals per sex per dose:

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

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1600, 5200 and 12000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report).
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ±20% of the mean for the appropriate sex and/or due to poor condition or ill health.
Other:
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random

Examinations

Maternal examinations:

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

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Mean daily consumption per animal (g/animal/day) was calculated for each phase
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Achieved dose was calculated using the mid-period body weight.
- Other: Food consumption was recorded for F0: Daily (including recovery phase). Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 19) but recommenced for males on Day 20. Reproductive females after mating food consumption was recorded daily until Day 12 of lactation.

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

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

Ovaries and uterine content:

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

Fetal examinations:

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

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

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

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

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

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

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

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

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

Indices:

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

Historical control data:

Historical control data was available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, overall body weight gain during the treatment period (Day 1 to 43) was significantly low (82% of control).
At the beginning of the recovery period the bodyweights for males previously treated at 12000 ppm were slightly lower than control (96%), however by Day 15 of recovery they had returned to similar levels (99% of control). There was evidence of recovery.
Females:
There was no effect on overall body weight gain (Day 1 to 43) in females of the toxicity phase.

Recovery phase females, receiving 12000 ppm during the treatment period, had similar body weights to Control at the start of the recovery phase; by the end of the recovery period, the treated females had markedly higher body weight gains (four-fold increase to Control).

At 12000 ppm, there was a mean weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15.
At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control.
At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental.

For females in gestation:
Receiving 12000 ppm had significantly low body weight at the start of gestation, mean gain over Days 0-7 was similar to Control. Their overall body weight gain (gestation day 0 to 20) was low (86%), when compared to control. Only the body weight change between Day 7 to 14 (82%) for females receiving 12000 ppm achieved statistical significance, however the change between Day 14 to 20 was also low (81%). Females at 1600 or 5200 ppm were unaffected during the gestation period.
For females in lactation:
Receiving 12000 ppm or 5200 ppm: overall body weight gain (1 to 13 of lactation) was low in females receiving, and this was largely influenced by the low body weight gain from Day 1 to 4 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Achieved doses generally maintained the intervals between dietary concentrations and increased during lactations due to increased physiological demand.
Males:
At 12000 ppm, food consumption was consistently lower than controls during the treatment period, particularly on Days 1 to 3 of treatment, although the extent of the difference diminished with time. During the recovery period, food consumption for males previously receiving 12000 ppm was slightly higher than control.
At 5200 ppm, males had lower intake on Day 1 with no consistent differences thereafter.
At 1600 ppm, no food consumption affects were observed.
Females:
At 12000 ppm, lower food intake in the first week of treatment achieving statistical significance at p < 0.01 level. During the recovery period, food consumption for females previously receiving 12000 ppm was generally slightly lower than in control.
At 5200 ppm, lower food intake was observed particularly on the first 2 days of treatment.
At 1600 ppm, lower food intake was observed particularly on the first 2 days of treatment.

For females in gestation:
Receiving 12000 ppm or 5200 ppm or 1600 ppm: food consumption tended to be marginally higher than control with once incidence of statistical significance.
For females in lactation:
Receiving 12000 ppm: food consumption was low from lactation Day 3 onwards
Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control. There was evidence of recovery.
Females:
At 12000 ppm, small but statistically significant decrease of hematocrit, hemoglobin concentration and erythrocyte count in males. With the exception to erythrocyte count of males, which did not achieve significance but was slightly low when compared to control.

However there were no significant differences from control of erythrocyte count in males, or of hematocrit and hemoglobin concentration in both male and female recovery animals. There was evidence of recovery.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, low aspartate aminotransferase, high glucose and low potassium concentration and low bile acids, when compared with control.
Low aspartate aminotransferase was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.
Females:
At 12000 ppm, low alkaline phosphatase or phosphorus concentrations and low bile acids and low glucose, when compared with control.
Low alkaline phosphatase or phosphorus was also seen at 5200 ppm and 1600 ppm. At the end of the recovery period there were no significant differences to control. There was evidence of recovery.

Urinalysis findings:

not examined

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, decreased terminal body weights were evident in males. Analysis of organ weights for the Toxicity phase animals revealed increased absolute and adjusted liver weights in all treated groups, with only males receiving 1600 ppm not achieving statistical significance. Recovery phase males previously treated at 12000 ppm had higher than control adjusted kidney weights, although this was considered as being reflected from a slightly lower than normal control value.
Females:
At 12000 ppm, Absolute and adjusted weights for the uterus, cervix and oviducts were low in all groups of treated toxicity females. This was not evident in reproductive phase females. It was considered as being reflected from a high control value. The difference in uterus, cervix and oviducts was not apparent in the reproductive phase females.
Absolute and adjusted ovary weights were low in reproductive phase females treated at 12000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no significant pathological findings observed that were considered related to treatment or were observed in the recovery phase groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 12000 ppm, Liver: Centrilobular hypertrophy. Thyroid: Follicular cell hypertrophy. Kidney: Accumulation of hyaline droplets
Females:
At 12000 ppm, Liver: Centrilobular hypertrophy.
There were no findings that were considered to be treatment related after 2 weeks of recovery.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

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

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

1. Thyroid Hormone Assessment:
Males:
At 12000 ppm, serum T4 levels were low in F0 males and slightly low in offspring. There was evidence of recovery.
At 5200 ppm, serum T4 levels were slightly low in F0 males. There was evidence of recovery.
During recovery, T4 concentrations in adult males returned to control levels.

There was considered to be no clear decrease seen T4 levels of offspring - review of the individual values did not reveal a convincing effect of treatment with mean hormone levels at 12000 ppm within the expected range.

There were no clear differences in Serum T3 levels in either the adult males or offspring on Day 13 of age; any differences from Controls were relatively minor, lacked relationship to dose and, therefore, attributed to normal biological variation.

No further analysis of T3/T4 or TSH was considered necessary.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were slightly low implantation counts in the females receiving 12000 ppm, compared to control and the historical control data range. It was considered that this reflected only one animal (An 0150) with only 4 implants.
By oversight, corpora lutea were not counted. However, in the absence of an effect of the number of implantations, this oversight had no effect on the study validity.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

It was considered offspring survival was not affected by treatment.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter size, offspring survival was not affected by treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation length and gestation index were unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females showed normal estrous cycles before treatment. Estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. Litter size, offspring survival was not affected by treatment.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 12 000 ppm

Based on:

test mat.

Remarks on result:

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

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 668 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

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

Maternal abnormalities

Abnormalities:

no effects observed

Description (incidence and severity):

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

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring body weight

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring survival

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring sex ratio

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Ano-genital distances for male or female offspring were unaffected by treatment with test item. There was higher incidence of litters/offspring containing males with nipples in the 12000 ppm group, when compared to control. The values were within the historical control data range.

Skeletal malformations:

no effects observed

Description (incidence and severity):

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

Visceral malformations:

no effects observed

Description (incidence and severity):

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

Other effects:

no effects observed

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 12 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

2

Key result

Dose descriptor:

NOAEL

Remarks:

gestation

Effect level:

>= 760 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

3

Key result

Dose descriptor:

NOAEL

Remarks:

lactation

Effect level:

>= 1 627 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

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

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity and for reproductive / developmental toxicity for males and females is considered to be 12000 ppm, which was equivalent to actual dose received: 760 mg/kg body weight per day (gestation) and 1627 mg/kg body weight per day (lactation).

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least six weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five female rats which received dietary test item at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. Ten males were treated in the 1600 and 5200 ppm doses for pairing purposes with the reproductive phase females. Recovery phase groups included five males and females treated at 0 ppm (Control) and 12000 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 1600, 5200 or 12000 ppm test item. 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 13 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. 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, untreated (no test item) diet, throughout the same relative treatment period. Mean achieved doses for males at 1600, 5200 or 12000 ppm were 93.7, 303 or 663 mg/kg/day, respectively. For females of the toxicity and recovery phase and reproductive phase females before pairing, mean achieved doses were 94.4, 296 or 668 mg/kg/day, respectively and ca. 100, 300 or 500-700 mg/kg/day for females before pairing. For females, mean achieved doses were 104, 322 and 760 mg/kg/day during gestation and 234.6, 747 or 1627 mg/kg/day during lactation at 1600, 5200 or 12000 ppm, respectively.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no effect on clinical condition, sensory reactivity and grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals.At 12000 ppm, there was a mean body weight loss over Days 1 to 8, with 7 animals showing a loss of 10 g or more; over Days 8 to 15, gain was similar to Control, although there was still a mean loss over Days 1 to 15. At 5200 ppm, mean weight gain over Days 1 to 8 was less than Control, although the difference was not statistically significant; over days 1 to 15 mean weight gain was marginally lower than Control. At 1600 ppm, slight differences were not obviously different from Control. There was no obvious explanation for the slight mean weight loss over Days 8 to 15, but in the absence of a similar finding at higher dose levels and in the absence of a lower gain over Days 1 to 15, this observation was considered to have been incidental. Body weight and food consumption during the gestation or lactation period was unaffected at 1600 or 5200 ppm, when taking into account the slight differences in litter size. For females at 12000 ppm, body weight gains during gestation or lactation were slightly lower than expected given the litter size and comparison between groups. Over the duration of the study, overall mean body weight gain was only lower in males at 12000 ppm. This coincides with the pattern of food intake seen in the earlier and later part of the study. Hematological investigations at the end of the treatment period revealed lower hematocrit, hemoglobin concentration and erythrocyte count in animals at 12000 ppm. There were no differences in these parameters following a 14 day recovery period. Blood chemistry investigations at the end of the treatment period revealed, in males, low aspartate aminotransferase in all treated groups, high glucose and potassium concentration at 12000 ppm and low bile acids at 12000 ppm. And for females, low alkaline phosphatase or phosphorus concentrations in all treated groups and low bile acids and glucose concentrations at 12000 ppm. There were no differences in these parameters following the 14 day recovery period.

 

At scheduled termination of the Toxicity phase animals after 6 weeks of treatment, decreased terminal body weights were evident in males treated at 12000 ppm. Analysis of organ weights for the Toxicity phase animals revealed high liver weights in all treated groups. After 14 days of recovery, Recovery phase males previously treated at 12000 ppm had high kidney weights. Microscopic changes related to treatment with the test item were seen in the liver of both sexes and the thyroid and kidneys of males. Centrilobular hepatocyte hypertrophy was seen in animals that received a dietary concentration of 12000 ppm. Follicular cell hypertrophy was seen in the thyroids of males that received a dietary concentration of 12000 ppm. Accumulation of hyaline droplets was seen in the kidneys of males that received a dietary concentration of 12000 ppm. There were no findings that were considered to be treatment related after 2 weeks of recovery. Levels of circulating thyroxine obtained from Main study male animals were low at 12000 ppm and further assessment of Recovery study males animals after a 2 week recovery period revealed no differences from Control after 2 weeks of no treatment. Analyses of samples for thyroxine (T4) obtained from F1 offspring on Day 13 of age were slightly low at 12000 or 5200 ppm. There were no differences from Control for levels of circulating Triiodothyronine (T3) in adult males or from F1 offspring on Day 13 of age. There was no effect of parental treatment on litter size, offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, nipple counts or macropathology. Macroscopic examination of offspring that died prior to scheduled termination or were killed at Day 13 of age did not reveal any findings that could be related to parental treatment.

 

Conclusion:

Dietary administration of test item to CD rats at concentrations up to and including 12000 ppm was generally well tolerated. Test item related histopathological changes were apparent in the kidney and thyroid of males and the liver of both sexes given 12000 ppm. The kidney changes detected in the males (hyaline droplets in tubules) were consistent with species-specific responses of the male rat in response to xenobiotics, and therefore may be of little value as an indicator of hazard to human health. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) in both sexes and associated thyroid changes (follicular cell hypertrophy) and lower T4 levels in males are deemed reversible. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was concluded to be 12000 ppm (equivalent to 663 mg/kg/day for males and 668 mg/kg/day for females), and the No Observed Adverse Effect Level (NOAEL) for the reproductive/developmental toxicity was concluded to be 12000 ppm (equivalent to 760 mg/kg/day during gestation and 1627 mg/kg/day during lactation).