Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity : target : (1RS,4Z,8E,12RS)-13-oxabicyclo[10.1.0]trideca-4,8-diene : Oral (dietary): NOAEL (rat): male/female: 3750 ppm (equivalent to ≥ 223 mg/kg bw/day), 2020

Read-Across: source data : 1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidized : Oral (dietary): NOAEL (rat): male/female: 3750 ppm (equivalent to ≥ 223 mg/kg bw/day), OECD TG 422, 2017

The changes in the thyroid glands observed in rodents were consequences of hepatic enzyme induction are of little toxicological relevance to man.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-04-2016 to 20-03-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: At ambient temperature in the dark
(ii) Formulated-diet: Formulations were prepared weekly and frozen in aliquots to allow daily feeding. Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable ; formulated-diet was prepared weekly.
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test. In the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 days when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure (full details available in the full study report). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

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

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

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

OTHER SPECIFICS: Not applicable.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 422 relevant guideline.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (full details reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: males ca. 71 days old ; females ca. 85 days old.
- Weight at study initiation: males 312 to 383 g ; females 229 to 299 g. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex.
- Fasting period before study: None
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. For acclimatisation pre-pairing, gestation, littering and lactation periods: Solid (polycarbonate) bottom cages were used. During pairing: Grid bottomed cages were used. These were suspended above absorbent paper which was changed daily. Cage enrichment and shelters was uses throughout the study except during pairing and lactation. Replaced as appropriate. Housing was group housing. With the number varying (single sex or mixed sex) during pre-pairing, pairing and after mating and gestation and lactation. Where females were specifically housed individually, or with litter.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum ; (removed overnight before blood sampling for hematology, blood chemistry, urine collection and adult thyroid hormone investigations.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: males: six days before commencement of treatment; females: twenty days before commencement of treatment.

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

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

IN-LIFE DATES: From: 2016-09-28 To: 2016-12-23
Route of administration:
oral: feed
Details on route of administration:
The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The required amount of test item was weighed into a suitable container. An amount of diet that approximately equalled the weight of test item was added and the mixture stirred together. A further amount of diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder, after which it was made up to the final weight of the premix with diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified).
- Storage temperature of food: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable ; formulated-diet was prepared weekly.
(ii) Formulated-diet: In a preceding in a (1) dietary formulation and method validation and (2) preliminary 14-day preliminary test. In the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for 40 days when formulations were stored frozen (-10 to -30°C) and up to 2 days when they were stored at ambient temperature (15 to 25°C). The diet was replaced daily during treatment/exposure (full details available in the full study report). (Stable for up to 40 days when stored frozen at 100 ppm and considered acceptable for 20000 ppm and at ambient for up to 30 hours for 100 ppm and 2 days at 20000 ppm).
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL dissolved into acetonee, within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report).
With LOD = 3.078 μg/mL and LOQ = 10.261 μg/mL; linearity = > 0.995 between 5 μg/mL and 25 μg/mL. Repeatability (n=6) of < 2%. Accuracy and precision was confirmed and mean procedural recovery was 93.78% (n=5 ; CV = 0.71%) at 100 ppm and 99.9% (CV = 1.95; n=5) at 20000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in SDS VRF-1 diet at nominal concentrations of 100 ppm and 20000 ppm. Storage was confirmed at ambient temperature (15 to 25°C) for up to 30 hours at 100 ppm and 2 days at 20000 ppm. At frozen temperature (-10 to -30°C) for up to 40 days. Frozen
stability at 20000 ppm was not assessed in error, however degradation and formulation instability is more pronounced at lower levels. As frozen stability was confirmed at 100 ppm for 40 days and all subsequent testing of achieved concentration samples were well within acceptable limits. It is considered that there is sufficient evidence to conclude test diets with a concentration of 20000 ppm are stable for 40 days under frozen conditions.
- Mean concentrations of diet-formulations analysed during the study were within ± 10/15% applied limits confirming accurate test item/diet formulation. Specifically, samples of each formulation prepared for administration in the first and last week of treatment were analysed for achieved concentration of the test item. The mean concentrations of test item were within 5% of nominal concentrations, confirming the accuracy of formulation. The difference from mean remained with 4%, confirming precise analysis.
Duration of treatment / exposure:
Toxicity phase and recovery groups: Two weeks pre-paring after minimum of 5 weeks treatment: as 6 weeks exposure and 2 week recovery period.
Reproductive phase groups: Minimum of two weeks of treatment and two weeks pairing and gestation until day 14 lactation.
Frequency of treatment:
Continuous via diet
Dose / conc.:
0 ppm
Remarks:
Control – Group 1; Basal diet
Dose / conc.:
417 ppm
Remarks:
Low – Group 2
Dose / conc.:
1 250 ppm
Remarks:
Intermediate – Group 3
Dose / conc.:
3 750 ppm
Remarks:
High – Group 4
Dose / conc.:
25.1 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 417 ppm group
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 1250 ppm group
Dose / conc.:
223 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for males for 3750 ppm group
Dose / conc.:
26.9 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 417 ppm (Main, Toxicity and Recovery phase groups)
Dose / conc.:
87 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 1250 ppm (Main, Toxicity and Recovery phase groups)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 3750 ppm (Main, Toxicity and Recovery phase groups)
Dose / conc.:
232 mg/kg bw/day (actual dose received)
Remarks:
Overall Mean for females for 3750 ppm during 6 weeks (Toxicity and Recovery phase animals and Main phase females for two weeks pre-pairing).
No. of animals per sex per dose:
Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
417 ppm (toxicity test) = 10
1250 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 5
3750 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
417 ppm (reproduction test) = 10
417 ppm (toxicity test) = 5
1250 ppm (reproduction test) = 10
1250 ppm (toxicity test) = 5
3750 ppm (reproduction test) = 10
3750 ppm (toxicity test) = 5
3750 ppm (recovery period) = 5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 417, 1250 and 3750 ppm) were chosen based upon the results obtained in a 14 day preliminary study (full details available in the full study report). Test item was exposed at dietary concentrations of 0 (control), 2500, 5000 and 7500 ppm respectively. It was determined that 7500 ppm would be unsuitable for a subsequent toxicity study. The overall mean achieved doses in animals receiving 2500, 5000 or 7500 ppm were 144, 304 and 470 mg/kg/day in males and 147, 322 and 490 mg/kg/day in females, respectively. There were no mortalities and no clinical signs related to treatment noted. No clear dose-related effect on food consumption or bodyweight gain was observed in males. Females receiving 7500 ppm initially lost weight and took 14 days to regain parity with their pre-treatment body weight. At 5000 ppm the females showed only 49% of the weight gain of the Control group over the 14-day exposure period, whilst at 2500 ppm the animals gained 86% of the Control weight gain. A non-dose related reduction in food consumption was seen in the females with a greater indication of a slight effect on food intake seen in animals receiving 5000 ppm than in animals receiving 7500 ppm. It is likely that these effects on body weight and food consumption were due to the reduced palatability of the formulated diets that was manifested in the females but not in the males. It was considered that this dietary concentration would be unsuitable for use in a study of longer duration. Water consumption was unaffected by treatment. There were no macroscopic findings that were considered to be related to treatment. High absolute and body weight adjusted liver weights were seen in males and females given 7500 ppm but with no macroscopic correlations.
- Rationale for animal assignment (if not random): Randomly assigned. Replacement animals were assigned (before treatment) based on variations on body weight ±20% of the mean for the appropriate sex and/or due to poor condition or ill health.
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): Random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during acclimatisation at least once daily ; during treatment at least twice daily.

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Mean daily consumption per animal (g/animal/day) was calculated for each phase
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes. Achieved dose was calculated using the mid-period body weight.
- Other: Food consumption was recorded for F0: Daily (including recovery phase). Food consumption was not recorded for males and females during the period when paired for mating (week 3) but recommenced for males in week 4. Reproductive females after mating food consumption was recorded daily until Day 13 of lactation.

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

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Peripheral blood samples: All toxicity groups at termination and recovery groups week 2. For reproductive phase: Day 14 of lactating (females) and test termination (males and females).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight, no more than 20 hours fasting)
- How many animals: five lowest numbered surviving toxicity males per group. All Toxicity phase females. All recovery males/females. All reproductive phase females.
- Parameters checked:
Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt). Additionally: Prothrombin time (PT) was assessed using IL PT Fibrinogen reagent and Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples: All toxicity groups at termination and recovery groups week 2. For reproductive phase: Day 14 of lactating (females) and test termination (males and females).
- Animals fasted: Yes (overnight, no more than 20 hours fasting)
- How many animals: five lowest numbered surviving toxicity males per group. All Toxicity phase females. All recovery males/females. All reproductive phase females.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein. Albumin/globulin ratio (A/G Ratio) (by calculation).

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

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

IMMUNOLOGY: No

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

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

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

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

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

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

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

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

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

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

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment related mortalities.

Group 1 male 36 was found dead during Week 7 of treatment. Prior to demise there had been no signs at routine physical examination and the individual was in good general condition. No macroscopic abnormalities were seen at necropsy. Following microscopic examination, no findings were seen that would have contributed to demise. Within the study the demise was not considered test item related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment.

Females:
Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Achieved doses generally maintained the intervals between dietary concentrations and increased during lactation due to increased physiological demand.

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

Females:
An initial reduction in food intake was observed in 3750 ppm group main study and toxicity/recovery phase females pf 89% of control. Following completion of treatment females that previously received 3750 ppm food consumption was high when compared with Controls; approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery.
Food efficiency:
not examined
Description (incidence and severity):
See 'Food consumption'
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water intake.
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology investigations revealed no treatment related changes.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were minor biochemical changes in blood plasma but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
At 3750 ppm: after 6 weeks of treatment males showed slightly elevated total protein. Males at both 1250 and 3750 ppm concentrations demonstrated low plasma bile acid concentration but without dose response. These findings were not evident in toxicity phase females after 6 weeks of treatment or in main study (reproductive) females on Day 14 of lactation. After two weeks without treatment dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls.
Females:
No effects.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were minor biochemical changes in urine but without toxicological significance, as the were minor without consistency between sexes and/or fully reversible.

Males:
After 6 weeks of treatment males at all dose levels showed low urinary specific gravity
At 3750 ppm: within males demonstrated increased urinary output when compared with Controls
Females:
At 3750 ppm: females demonstrated high urinary pH with Controls
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity and hindlimb grip strength, motor activity scores and arena observations appeared unaffected by treatment.

Any observations appeared isolated, or when with inter-group statistical significance: without dose-response and/or within control ranges and/or historic control ranges.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males:
At 3750 ppm: after 6 weeks of treatment males showed low adjusted mean heart weight (p<0.05) and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). There were no correlating micropathological/histopathological findings to account these differences compared to Controls. In addition the adjusted epididymal and glans penis weights were low but these differences are not considered to be of biological significance. After two weeks of recovery adjusted epididymal, glans penis, heart, adrenal and thyroid weights for males that received 3750 ppm were not significantly different from Controls.
Thymus weights after two weeks of recovery for males that received 3750 ppm were significantly low when compared with Controls; in the absence of an effect after 6 weeks of treatment or in the recovery phase females this is considered not to be of toxicological significance.

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

See: “histopathology : non-neoplastic” for further comments, regarding lack of biological significance and/or relevance to man.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant macroscopic/pathological findings observed that were considered related to treatment or were observed in the treatment and recovery phase groups.

On Day 14 of lactation pale areas were seen on the liver of one female at 1250 ppm and one female at 3750 ppm. In addition depressions were seen in the corpus region of the stomach of one female at 417 ppm and one female at 3750 ppm.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males:
At 3750 ppm: Microscopic examination performed after 6 weeks of treatment revealed:
LIVER: minimal centrilobular hepatocyte hypertrophy. Following the 2 week recovery period, centrilobular hepatocyte hypertrophy was seen in two males (m21 and m25) and in one female (f104) previously treated with 3750 ppm for 6 weeks and therefore demonstrated partial and near full recovery in males and females respectively.
THYROIDS: follicular cell hypertrophy was seen in males (m22 and m23) and females treated, and in a few males treated with 417 or 1250 ppm. This hypertrophy contributed to the increased body weight adjusted group mean combined thyroid and parathyroid weights in males treated with 3750 ppm, compared with male controls. Following the 2 week recovery period, follicular cell hypertrophy was seen in two males previously treated with 3750 ppm for 6 weeks. Therefore, in males, the relevant incidence in main study and recovery phase animals was the same (40%) and so recovery had not occurred but complete recovery was seen in females.

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

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

Incidental Findings
THYMUS : minimal or slight involution/atrophy was seen in a few control females and females treated with 3750 ppm. As this change was not seen in any unmated control females or females treated with 3750 ppm for 6 weeks, it was considered that the involution/atrophy was related to the ‘stress’ of pregnancy and lactation within rodents and therefore unrelated to treatment (literature citations given in the full study report). The involution/atrophy was considered to have contributed to the recorded decreased body weight adjusted group mean thymus weights in females treated with 3750 ppm, compared with female controls.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected. There were no microscopic neoplastic findings that were considered to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
1. Thyroid Hormone Assessment:
F0 Males: analyses of samples for thyroxine (T4) did not reveal any differences that could be attributed to treatment, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary.
F1 Female offspring: analyses of samples for thyroxine (T4) did not reveal any differences that could be attributed to treatment, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 750 ppm
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 223 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 750 ppm
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males is considered to be 3750 ppm which was equivalent to actual dose received: 223 mg/kg body weight per day. The no-observed-adverse-effect level (NOAEL) for females is considered to be 3750 ppm which was equivalent to actual dose received: 225 mg/kg bw/day. The test item related histopathological changes in the kidneys of males, were considered within the study to be non-adverse (and/or would be adaptive changes). Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, It was considered that this was a species specific observation and is not toxicologically significant in man.
Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least six weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five or ten female rats which received dietary test item at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Five females were treated in the 0 (control) and 3750 ppm doses for toxicity but not used pairing purposes with the reproductive phase males. Recovery phase groups included five males and females treated at 0 ppm (Control) and 3750 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Main study males were treated for two weeks before pairing up to necropsy after at least six weeks. Toxicity phase females were treated for at least six weeks. Main study males were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 offspring received no direct administration of the test item; any exposure was in utero or via the milk prior to termination. A similarly constituted Control group was assigned to each F0 phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 417, 1250 or 3750 ppm for the toxicity and recovery phases were 25.1, 75.0 and 223 mg/kg bw/day in males and 0 and 232 mg/kg bw/day in 0 (control) and 3750 ppm females, respectively. Mean achieved doses at 417, 1250 or 3750 ppm for the main study (reproductive phase), toxicity and recovery females was: 26.9, 75.6 and 225 mg/kg bw/day. The mean achieved dose during gestation in main study females was: 28.7, 87.0 and 259 mg/kg bw/day and during lactation was: 67.3, 192 and 598 mg/kg bw/day. Mean achieved doses were higher due to the increased physiological demand of lactation.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no treatment-related effect on clinical condition, sensory reactivity and (hind/fore limb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. There was no effect on water intake, and no toxicologically significant changes in haematological parameters. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult Toxicity phase males or in the Day 13 male and female offspring, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary. During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm indicated body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males at 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery). Males indicated food consumption that was similar to concurrent Controls. Blood chemistry investigations after 6 weeks of treatment males at 3750 ppm demonstrated slightly elevated total protein and at 1250 and 3750 ppm, low plasma bile acid concentration without dose-response. These findings were not evident in toxicity phase females or in main study females on Day 14 of lactation. After two weeks recovery the total protein, albumin concentration and albumin/globulin ratio for recovery phase males at 3750 ppm was comparable with Controls. Recovery was therefore evident. Urinalysis investigations performed after 6 weeks of treatment to males at all dose levels indicated low urinary specific gravity, whereas at 3750 ppm males had increased urinary output and females high urinary pH when compared with Control values. After two weeks of recovery males at 3750 ppm had urinary output and specific gravity that were essentially comparable with Controls.Males that received 3750 ppm had low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery for males : adjusted heart, adrenal and thyroid weights were not significantly different from Controls. Toxicity phase females at 3750 ppm had significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; approximately 115% of Controls). At 3750 ppm, on Day 14 of lactation females had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls. Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

 

Conclusion:

Dietary administration of test item to Sprague-Dawly rats for 6 weeks at concentrations up to and including 3750 ppm was generally well tolerated by the Toxicity phase males/females, and Reproductive phase females and their offspring. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3750 ppm (mean achieved dose males/females 223 mg/kg bw/day) and/or reproductive/developmental toxicity at 3750 ppm (mean achieved doses females; 259 mg/kg bw/day during gestation and 598 mg/kg bw/day during lactation). Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man. Based on these findings, the NOAEL for general systemic toxicity via the dietary route was concluded to be 3750 ppm (mean achieved dose: 223 mg/kg bw/day) in male/female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
223 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable.

Additional information

Repeated dose - Oral:

Key Study : OECD TG 422, 2017 : Read-Across SOURCE ( 1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidized ) : The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by dietary administration for at least six weeks with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicology treatment groups with a control was conducted, each comprising five or ten male and five or ten female rats which received dietary test item at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Five females were treated in the 0 (control) and 3750 ppm doses for toxicity but not used pairing purposes with the reproductive phase males. Recovery phase groups included five males and females treated at 0 ppm (Control) and 3750 ppm. Reproductive phase females, ten per group (10) were treated at doses of 0 (Control), 417, 1250 or 3750 ppm test item. Main study males were treated for two weeks before pairing up to necropsy after at least six weeks. Toxicity phase females were treated for at least six weeks. Main study males were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 offspring received no direct administration of the test item; any exposure was in utero or via the milk prior to termination. A similarly constituted Control group was assigned to each F0 phase, and received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 417, 1250 or 3750 ppm for the toxicity and recovery phases were 25.1, 75.0 and 223 mg/kg bw/day in males and 0 and 232 mg/kg bw/day in 0 (control) and 3750 ppm females, respectively. Mean achieved doses at 417, 1250 or 3750 ppm for the main study (reproductive phase), toxicity and recovery females was: 26.9, 75.6 and 225 mg/kg bw/day. The mean achieved dose during gestation in main study females was: 28.7, 87.0 and 259 mg/kg bw/day and during lactation was: 67.3, 192 and 598 mg/kg bw/day. Mean achieved doses were higher due to the increased physiological demand of lactation.

 

There were no treatment-related premature mortality among adult animals during the course of the study. There was no treatment-related effect on clinical condition, sensory reactivity and (hind/fore limb) grip strength, motor activity, estrous cycles, pre-coital interval, mating performance, fertility and gestation length or macropathology of the adult animals. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. There was no effect on water intake, and no toxicologically significant changes in haematological parameters. There was no effect of treatment on the circulating levels of thyroxine (T4) in adult Toxicity phase males or in the Day 13 male and female offspring, therefore further assessment of T3/T4 and thyroid stimulating hormone (TSH) was not considered necessary. During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm indicated body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males at 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery). Males indicated food consumption that was similar to concurrent Controls. Blood chemistry investigations after 6 weeks of treatment males at 3750 ppm demonstrated slightly elevated total protein and at 1250 and 3750 ppm, low plasma bile acid concentration without dose-response. These findings were not evident in toxicity phase females or in main study females on Day 14 of lactation. After two weeks recovery the total protein, albumin concentration and albumin/globulin ratio for recovery phase males at 3750 ppm was comparable with Controls. Recovery was therefore evident. Urinalysis investigations performed after 6 weeks of treatment to males at all dose levels indicated low urinary specific gravity, whereas at 3750 ppm males had increased urinary output and females high urinary pH when compared with Control values. After two weeks of recovery males at 3750 ppm had urinary output and specific gravity that were essentially comparable with Controls.Males that received 3750 ppm had low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery for males : adjusted heart, adrenal and thyroid weights were not significantly different from Controls. Toxicity phase females at 3750 ppm had significantly high adjusted mean liver weight when compared with Controls (p<0.01 ; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05 ; approximately 115% of Controls). At 3750 ppm, on Day 14 of lactation females had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls. Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

 

Conclusion:

Dietary administration of test item to Sprague-Dawly rats for 6 weeks at concentrations up to and including 3750 ppm was generally well tolerated by the Toxicity phase males/females, and Reproductive phase females and their offspring. It was therefore concluded that within the context of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3750 ppm (mean achieved dose males/females 223 mg/kg bw/day) and/or reproductive/developmental toxicity at 3750 ppm (mean achieved doses females; 259 mg/kg bw/day during gestation and 598 mg/kg bw/day during lactation). Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Centrilobular hepatocyte hypertrophy is an indication of enzyme induction and follicular cell hypertrophy is an indicator of increased thyroid hormone synthesis. As hepatic enzymes have a recognised role in thyroid hormone metabolism and clearance, an increase in enzyme activity can result in increased compensatory thyroid hormone production. The findings in this study therefore demonstrate that the changes in the thyroid glands were consequences of hepatic enzyme induction and consequential disruption of the normal feedback control of the thyroid glands. Thyroid changes observed in this study are of little relevance to man since the hormone binding profiles differ and the rate of metabolic clearance of thyroxine is much slower in man than in rats and, consequently, the thyroid gland in man is not as markedly influenced by hepatic enzyme induction. It was therefore considered, the effects were non-adverse (adaptive effects) and/or of little toxicological relevance to man. Based on these findings, the NOAEL for general systemic toxicity via the dietary route was concluded to be 3750 ppm (mean achieved dose: 223 mg/kg bw/day) in male/female rats.

Justification for classification or non-classification

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

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

References:

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