Acetic acid, anhydride, reaction products with 1,5,10-trimethyl-1,5,9-cyclododecatriene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2020 - Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-330-9

EC Name:

-

Cas Number:

144020-22-4

Molecular formula:

C17H26O

IUPAC Name:

1-[(1E,5Z,9Z)-2,5,10-trimethylcyclododeca-1,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one; 1-[(1S)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1S)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 68 to 74 days for males and 103 to 110 days for females
- Weight at study initiation: 301 to 377 g for males and 215 to 287 g for females
- Housing: Cages comprised of a polycarbonate body with a stainless-steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood-based bark-free fibre bedding, which was changed at appropriate intervals each week. Number of animals per cage: Pre-pairing, up to four animals of one seks. Pairing, one male and one female. Males after mating, up to four animals. Gestation, one female. Lactation, one female + litter. Environmental enrichment: Aspen chew block, A soft white untreated wood block; provided to each cage throughout the study (except during late gestation and lactation) and replaced when necessary. Plastic shelter, provided to each cage throughout the study (except during pairing, late gestation and lactation) and replaced at the same time as the cages. Paper shavings Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.
- Diet (ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Females: At least 5 days before commencement of oestrous cycle evaluation. Males: At least 5 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Supplier’s analytical certificates are scrutinized and approved before any batch of diet is released for use. Certificates of analysis are routinely received from the water supplier. Bottles were changed at appropriate intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): filtered, not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 August 2020 (treatment commenced) To: 01 October 2020 (last female necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was weighed. Where appropriate, starting with the lowest concentration, approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until humongous. A series of formulations at the required concentrations were prepared in ascending order.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly and prepared in advance of the first day of use.
- Storage temperature of food: Refrigerated (2 to 8°C).

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.

Analytical verification of doses or concentrations:

yes

Remarks:

Achieved concentration was analysed from the first and last formulation preparations.

Details on analytical verification of doses or concentrations:

The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis. Procedural recoveries remained within the range established during the validation, confirming the continued accuracy of the analytical procedure. The homogeneity and stability of formulations during storage was determined as part of another study, Study Number IFF 0369/073192. Formulations were stable for 48 hours at ambient (15 to 25°C) and 8 days at refrigerated (2 to 8°C) temperatures.

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 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Findings of a previous OECD 407 study (IFF 0369/073192). At 1000 mg/kg/day several effects were observed, and the NOAEL was determined to be 150 mg/kg/day. Based on this data, a high dose of 500 mg/kg/day was chosen for investigation on this study with the expectation that liver weights would be significantly increased. Low and intermediate dose levels had been selected at 50 and 150 mg/kg/day, respectively.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for evidence of ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: F0 males: Week 1 - daily, Week 2 to 4 - twice weekly, Week 5 onwards – once each week. F0 females: Week 1 – daily, Week 2 – twice during the week, Gestation phase – Days 0, 7, 14 and 20, Lactation Phase – Days 1, 6 and 12. Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, one to two hours after completion of dosing and as late as possible in the working day. Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: Before dosing on the day that treatment commenced (Day 1) and weekly thereafter and on the day of necropsy. F0 females Before dosing on the day that treatment commenced (Day 1) and weekly before pairing. Days 0, 7, 14 and 20 after mating, Days 1, 4, 7 and 13 of lactation and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly or daily diet consumption calculated as g food/animal/week or g food/animal/day: Yes
Weekly, from the day that treatment commenced. 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. For females after mating food consumption was performed to match the body weight recording: Days 0-7, 7-14, 14-20 after mating. Days 1-4, 4-7 and 7-13 of lactation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (light general anesthesia induced by isoflurane)
- Animals fasted: Not specified
- How many animals: 5 / sex / group (five lowest numbered surviving males and first five lactating females with a surviving litter)
- Parameters examined: 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), and Platelet count (Plt). Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
*derived values calculated in ClinAxys

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Not specified
- How many animals: 5 / sex / group (five lowest numbered surviving males and first five lactating females with a surviving litter)
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Bile acids (BiAc), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).

SERUM HORMONES: Yes, T4 and TSH
- Time of blood sample collection: F0 males and females at termination,
- Animals fasted: No
- How many animals: F0 - all surviving males and all surviving reproductive phase females.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 5 of treatment for males and at Day 7-9 of lactation for females
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (approach response, pinna reflex, auditory startle reflex and tail pinch response), grip strength and motor activity

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

Dry smears: For 15 days before pairing using cotton swabs
Wet smears: Using pipette lavage during the following phases: For 14 days before treatment (all females including spares); animals that fail to exhibit 4-5 day cycles were not allocated to study. After pairing until mating. For four days before scheduled termination.

Sperm parameters (parental animals):

- testis weight and epididymis weight was examined
- 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:

PARAMETERS EXAMINED
- Clinical observations: 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, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
- Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
- Individual offspring body weights: Days 1, 4, 7, 11 and 13 of age.
- Ano-genital distance: Day 1 - all F1 offspring.
- Nipple/areolae count: Day 13 of age - male offspring.
Thyroid Hormone Analysis: Day 4 of age Offspring: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation), one for T4 (serum) and one for TSH (serum). Day 13 of age F1 offspring, two males and two females per litter (where possible), two for T4 (serum); where possible one male and one female and two for TSH (serum); where possible one male and one female.
GROSS EXAMINATION OF DEAD PUPS:
In case of premature deaths where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained.
Offspring at scheduled termination: All 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, where possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

All adult animals were subject to a detailed necropsy.
Time of Necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to mate: Day 25 after last day of pairing.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 13 of lactation.
F1 offspring: Day 13 of age.

GROSS PATHOLOGY: Yes
- Organs weighed for the five lowest numbered surviving males and females with a surviving litter per group at scheduled termination, and all F0 adult decedents: adrenals, brain, caecum, epididymides, heart, kidneys, liver, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus
- Organs weighed for remaining F0 males and females per group: epididymides, liver and testes
- Females number of implantation sites for each uterine horn was counted and confirmed if none were visible at visual inspection
- Mammary tissue appearance of females whose litter died before day 13 of lactation
- Offspring in case of premature deaths where possible a fresh macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained.
- Offspring at scheduled termination were all 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, where possible.

HISTOPATHOLOGY: Yes
- The five lowest numbered surviving males and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination: abnormalities, adrenals, brain, caecum, colon, duodenum, epididymides, eyes, heart, ileum, jejenum, kidneys, liver, lungs, lymph nodes, ovaries, peyer’s patch, prostate, rectum, sciatic nerve, seminal vesicles with coafulating glands, skeletal muscle, skin with mammary glands, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, thrachea, urinary bladder, uterus, vagina.
- all males of groups 2 and 3: liver and thyroid
- all F0 animals: abnormalities only

Postmortem examinations (offspring):

In case of premature deaths where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained.
Offspring at scheduled termination: All 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, where possible.

Statistics:

- For grip strength, motor activity, body weight, food consumption, implantations, litter size, sex ratio percentage male, post implantation survival index, ano-genital distance, organ weight and clinical pathology data: Parametric analysis if Bartlett’s test was not significant at the 1% level, followed by t-tests or F1 approximate test, followed by Willams’ test or Dunnet’s test. Non-parametric analysis if Bartlett’s test was significant at the 1% level, followed by Kruskal-Wallis’ test and Wilcoxon or by H1 approximate test followed by Shirley’s test or Steel’s test.
- For grip strength, motor activity and clinical pathology data: if 75% of the data were the same value Fischer’s exact tests were performed
- For live birth and viability indices: if the Cochran-Armitage test was significant at the 5% level one-tailed step-down testing was performed, if it was not significant a Chi-square test was applied. If the latter was significant at the 5% level Fisher’s exact test were performed.
- For gestation length an exact two-tailed Linear-by-linear test was applied.
- For oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was applied.
- For number conceiving and number fertile an exact one-tailed (lower-tail) Cochran-Armitage test was applied.
- For organ weight data analysis of covariance was performed using terminal body weight as covariate.
- For the litter average ano-genital distance, analysis of covariance was performed using the average pup bodyweight for each litter as the covariate.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or
1% (p<0.01) level. Analysis used are appropriate.

Reproductive indices:

Pre-coital interval, Percentage mating, Conception rate, Fertility index, Gestation index, Litter size.

Offspring viability indices:

Post-implantation survival index, Live birth index, Viability index, Lactation index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs or signs observed following dose administration were observed for either sex pre- or post-pairing or during gestation and lactation for females.

For more details see Tables 11.1, 11.2, 11.3, and Appendix 12.1. 12.2 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female that received 150 mg/kg/day (animal 3F 102) was despatched for welfare reasons on Day 24 of gestation due to signs of piloerection, whole body pallor, pale eyes, and red discharge in the vaginal area. Macroscopic examination revealed abnormal dark contents in the cecum, colon, duodenum, ileum, jejunum, stomach and vagina, a cyst on the left kidney and many organs were pale in colour. Microscopic examination revealed a mild hemorrhage in the vaginal lumen. This female was found to be pregnant with 8 implantations and the condition of the animal suggests a possible problem with parturition associated with the extended gestation period for this animal. Given the single incidence in the mid-dose group, this premature death was not considered to be related to treatment.

For more details see Attachment 13.5 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males that received 500 mg/kg/day, mean body weight gain during the first week of treatment was 56% lower than control, with differences attaining statistical significance. Weekly body weight gains remained lower, but not statistically significant, throughout the remainder of the study resulting in statistically significantly lower overall body weight gain (Days 1-43; 68% of Controls) at the end of the study.

For males that received 150 or 50 mg/kg/day, mean body weight gains during the first week of treatment were 16 and 12% lower than the control, respectively, but failed to achieve statistical significance. There was no consistent pattern for weekly body weight gain observed during the remainder of the study, although gains were occasionally lower than control, and overall body overall body weight gain (Days 1-43; 90% and 85% of Controls at 150 or 50 mg/kg/day, respectively) was only marginally lower than Control at the end of the study.

Prior to pairing, in all groups of treated females, body weight gain was slightly lower than Control during the second week of treatment (51%, 95% and 90% of Controls at 500, 150 and 50 mg/kg/day respectively) but with no dose response apparent. Females that received 500 mg/kg/day had slightly lower overall body weight gain (110g; 90% of Controls) during gestation, however, statistical significance was not attained. At 500 mg/kg/day, group mean absolute body weight was 12% lower on Day 1 of lactation and this trend continued throughout lactation, however, although a marginal mean body weight loss was observed initially (Days 1-4), overall weight gain was similar to Controls. There was no effect of treatment on female body weight gain at 150 or 50 mg/kg/day during gestation or lactation.

For more details see Figure 10.3, 10.4, and 10.5, 10.6, Table 11.8, 11.9, and 11.10, Appendix 12.7, 12.8, and 12.9 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intake for all groups of treated males during the first week of treatment was lower than Control, with a dose response apparent (11, 6, and 4% at 500, 150, and 50 mg/kg/day, respectively), and remained 5% lower for males at 500 mg/kg/day in Week 2. After pairing, food intake for all groups of treated males was similar to Control.

For females at 500 mg/kg/day, food intake before pairing was 6 and 7% lower than Control in Week 1 and 2, respectively, and this trend continued throughout gestation and lactation, although statistical significance was not attained. There was no effect of treatment on female food intake at 150 or 50 mg/kg/day prior to pairing or throughout gestation and lactation.

For more details see Table 11.11, 11.12, and 11.13, Appendix 12.10, 12.11, and 12.12 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology investigation at scheduled termination of males and females revealed no conclusive treatment-related effects.

Statistically significantly lower mean erythrocyte count (7.0%), haematocrit (4.5%) and hemoglobin concentration (5.2%) and higher mean absolute reticulocyte count (33.3%), compared to Control, were apparent for males that received 500 mg/kg/day. For females that received 500 or 150 mg/kg/day, mean erythrocyte count (5.4 and 7.6%, respectively) and haematocrit (5.1 and 6.3%, respectively) were also statistically significantly lower than Control, but there was no effect on hemoglobin concentration or mean absolute reticulocyte count.

Statistically significantly lower mean total leucocyte count, compared to Control, was apparent for both sexes that received 500 mg/kg/day (34.7 and 39.9% for male and females, respectively), principally due to statistically significantly lower mean numbers of lymphocytes (37.9 and 49.6%, respectively). Mean lymphocyte counts were also statistically significantly lower than Control for females that received 150 or 50 mg/kg/day (29.4 and 32.7%, respectively). Statistically significantly lower mean basophil counts were apparent for females that received 150 or 50 mg/kg/day (60% at both dose levels) and both sexes that received 500 mg/kg/day (57 and 60% for males and females, respectively) compared to control, although inter-group differences were not convincing. The mean number of large unstained cell counts was statistically significantly lower than Control for treated males (73.3, 46.7, and 73.3% at 500, 150, and 50 mg/kg/day, respectively) but values showed no clear dose-relationship and no similar change was apparent in females.

For more details see Table 11.20, Appendix 12.14 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations at scheduled termination of males revealed slightly but statistically significantly higher mean calcium levels, compared to Control, for males that received 500 or 150 mg/kg/day (4.7 and 3.5%) however, mean values for other blood electrolytes were similar to Control.

Statistically significantly lower mean creatinine (23.8%) and glucose levels (15.6%), compared to Control, were also apparent for males that received 500 mg/kg/day. These males also showed higher mean albumin (5.6%) and albumin/globulin ratio (11.8%), although the level of mean total protein appeared unaffected. No similar statistically significant differences were observed in the females.

A statistically significantly 15.6% lower mean aspartate aminotransferase (AST) activity was apparent for males that received 500 mg/kg/day but the decrease in the circulating level of this intra-cellular enzyme is unlikely to be of any toxicological significance. No similar statistically significant differences were observed in the females.

All other differences from Control in males and females were generally small, confined to one sex, or the magnitudes were not dose-related and consequently were considered to represent normal biological variation.

For more details see Table 11.21, Appendix 12.15 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum Thyroxine (T4) concentrations for all groups of treated F0 adult males at termination were higher than the mean T4 concentration observed in the respective Control group with a dose response apparent (21, 21, and 11% increase at 500, 150, and 50 mg/kg/day, respectively) and statistical significance attained at 500 and 150 mg/kg/day.

As a result of this finding, the Thyroid Stimulating Hormone (TSH) serum samples were analysed from the F0 adult males from all groups. A statistically significant 73% increase in mean serum TSH levels was observed, compared to Control, in F0 males that received 500 mg/kg/day. There was no effect of treatment on mean serum TSH levels in F0 males that received 150 or 50 mg/kg/day.

For more details see Attachment 13.3 and 13.1 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Reactivity Observations and Grip Strength (Table 11.4, 11.5, Appendix 12.3, 12.4, Attachment 13.5 of the attached report)
- No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 5 of treatment for males or during Days 7-9 of lactation for females.

Motor Activity (Figure 10.1, 10.2, Table 11.6, 11.7, Appendix 12.5, 12.6, Attachment 13.5 of the attached study report)
- Motor activity assessment of males during Week 5 of treatment and for females during Days 7-9 of lactation revealed no conclusive treatment-related effects.
- For females that received 500 mg/kg/day, mean total low beam activity, compared to Control, was statistically significantly 40% lower over the 60-minute assessment, principally due to lower mean activity during the start of the recording period. Mean total high beam activity was 38% lower than Control for these females but did not achieve statistical significance.
- For females that received 150 mg/kg/day, mean low beam activity, compared to Control, was statistically significantly 30% lower during the first 6-minute recording interval, however overall activity over the 60-minute interval, although 28% lower than Control, did not achieve statistical significance. Total high bean activity for these females was similar to Control.
- For both sexes that received 50 mg/kg/day and for males that received 500 or 150 mg/kg/day mean low beam and high beam activity over the 60-minute assessments appeared similar to Control in the OECD 422 study record under IUCLID chapter 7.5.1.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At terminal sacrifice, Trimofix-related microscopic findings were noted in the liver and thyroid. Minimal to slight centrilobular hepatocyte hypertrophy was noted in four out of five males that received 500 mg/kg/day and in one out of five males that received 150 mg/kg/day. Minimal follicular hypertrophy was noted in the thyroid of four out of five males that received 500 mg/kg/day and in two out of five males that received 150 mg/kg/day.

The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent Controls), and/or their severity was as expected for Sprague-Dawley rats at this age; therefore, they were considered not test article related.

For more details see Attachment 13.4 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There was an increase in the number of females (20-30%) showing irregular cycle length during the pre-pairing treatment period compared to the incidence recorded in the pre-treatment period, however as all groups, including Control, were affected and there was no subsequent effect on pre-coital interval or pregnancy rate, this finding was considered to be of no biological significance.

For more details see Table 11.14, 11.15, 11.16, 11.17, 11.18, 11.19, Appendix 12.13, Attachment 13.5 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was a tendency towards longer gestation length (0.5 days), compared to Control, for females that received 500 mg/kg/day however, this was not associated with any effect on parturition/littering and there was no effect of treatment on gestation index; most pregnant females at this dose level successfully gave birth to a live litter.

For more details see Table 11.14, 11.15, 11.16, 11.17, 11.18, 11.19, Appendix 12.13, Attachment 13.5 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs amongst the offspring.

For more details see Appendix 12.16 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of implantations, post implantation survival index (%), post-natal litter size, viability index (%) on Day 4 and the sex ratio (% males) were all considered to be unaffected by maternal treatment with Trimofix.

Live birth index (%) was 12.3%, but not statistically significantly, lower than Control at 500 mg/kg/day due to the total litter loss in this group.

For more details see Table 11.22, 11.23 and 11.24, Appendix 12.17, 12.18 and 12.19 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weight on Day 1 of age was 6 and 7.8% lower than Control for male and female offspring, respectively, of females that received 500 mg/kg/day. Subsequent absolute group mean body weights and body weight gains of these offspring (both sexes) were slightly lower, compared with Control, and resulted in lower overall body weight gain (Day 1 to 13 of age) (16.2 and 16.8% lower compared to control for male and female pups, respectively) which attained statistical significance for the female offspring.

There was no effect of treatment on offspring body weights at 150 or 50 mg/kg/day.

For more details see Figure 10.7, 10.8, Table 11.26, Appendix 12.21 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum T4 concentrations for male and female offspring on Day 13 of age derived from treated parents were higher (31, 28, and 26% for male and 14, 18, and 10% for female rats at 500, 150, and 50 mg/kg/day, respectively) than the mean T4 concentrations found in the respective Control offspring, however statistical significance was not attained at any dose level, no dose-relationship was apparent for the female offspring and all group mean values were within the historical control data (HCD) range. Therefore, these findings were considered to represent normal biological variation and no further investigation of TSH samples was conducted.

For more details see Attachment 13.3 and 13.1 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on male or female offspring ano-genital distance at any dose level investigated.

For more details see Table 11.25 and Appendix 12.20 of the attached study report.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

One male offspring derived from parents that received 150 mg/kg/day was observed with 1 nipple on Day 13 of age and 2 male offspring (2 litters affected) derived from parents that received 50 mg/kg/day were observed with 1 nipple each on Day 13 of age. Given the low incidence and since no male offspring derived from parents that received 500 mg/kg/day were observed with nipples, the presence of these nipples was considered unrelated to treatment.

For more details see Appendix 12.22 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related macroscopic observations were present in the offspring of treated animals killed or dying before scheduled termination or at scheduled termination.

For more details see Attachment 13.5 of the attached study report in the OECD 422 study record under IUCLID chapter 7.5.1.

Details on results (F1)

Three Control females (Nos. 1F 111, 114 and 141) and one female (No. 4F 128) that received 500 mg/kg/day failed to litter and were dispatched to necropsy on Day 25 after mating. Of these animals, two Control females (Nos. 1F 111 and 141) and one female (No. 4F 128) that received 500 mg/kg/day were found to be not pregnant and one Control female (No. 1F 114) was pregnant with 14 implantations. Control females 1F 114 and 141, both showed adhesions in the thoracic cavity with female 1F 114 also showing masses affecting multiple organs at macroscopic necropsy. Microscopic evaluation revealed multifocal granulomatous foci associated with foreign material, observed in lungs and pleura; collectively these finding indicated accidental trauma associated with the dosing procedure. These findings possibly contributed to the failure of these animals to achieve pregnancy or litter. There were no obvious macroscopic findings observed for females 1F 111 or 4F 128 (or their respective male partners) that could explain why these females failed to achieve pregnancy and no relationship to treatment was inferred. Furthermore, one female (No. 4F 123) that received 500 mg/kg/day was dispatched to necropsy on Day 1 of lactation with a total litter loss.

Therefore, taking into account the premature death at 150 mg/kg/day, 7, 10, 9 and 8 litters for Groups 1, 2, 3 and 4, respectively, were available to assess litter size, sex ratio, survival indices and body weights (F1).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD TG 422, GLP), the systemic NOAEL was determined to be 150 and mg/kg bw based on the increase of liver weights effect. The fertility NOAEL is >= 500 mg/kg bw based on the absence of an adverse effect. The developmental NOAEL is >= 500 mg/kg bw based on the absence of an adverse effect.

Executive summary:

This executive summary is the same as presented in the repeated doses toxicity section.

In a combined 28-days repeated dose toxicity study with reproduction/developmental screening performed in accordance with OECD TG 422 and GLP, the substance was administrated via oral gavage to Sprague-Dawley rats (10 per dose per sex) at dosages of 50, 150 and 500 mg test item/kg bw/day. Control animals received the vehicle, corn oil, alone. All parameters measured from the OECD TG 422 have been recorded.

Clinical signs: No adverse effects considered to be associated with treatment were observed for mortality, clinical signs, neurobehavior, bodyweight and food consumption.

Haematology: No adverse effects considered to be associated with treatment were observed.

Biochemical parameters: No adverse effects considered to be associated with treatment were observed.

Organ effects weight: Absolute and body weight adjusted liver weights were increased in all groups of treated males with a dose response apparent and statistical significance attained at 500 and 150 mg/kg/day for the adjusted values. Compared to Control, absolute and adjusted kidney weights were also increased in all groups of treated males with statistical significance attained for the adjusted values at 500 mg/kg/day, but there was no dose response apparent. On Day 13 of lactation, absolute and body weight adjusted liver weights for females that received 500 or 150 mg/kg/day were higher with statistical significance attained at 500 mg/kg/day for adjusted weights. Absolute and body weight adjusted spleen weights were lower in all treated females, statistical significance was attained for all treated groups for the adjusted weights.

Macroscopy and histopathology: No adverse effects considered to be associated with treatment were observed.

Fertility: No adverse effects considered to be associated with treatment were observed.

Development: No adverse effects considered to be associated with treatment were observed.

Under the conditions of this study, the NOAEL for repeated dose toxicity was established to be 150 mg/kg bw/day for males and females, based on the increase in liver weights effect. The fertility NOAEL is >= 500 mg/kg bw based on absence of reproductive toxicity up to the highest dose level tested. The developmental NOAEL is >= 500mg/kg bw based on absence of developmental toxicity up to the highest dose level tested. Based on the results of the available repeated dose studies the substance does not have to be classified for reproductive toxicity (fertility and developmental toxicity) according to EU CLP (EC 1272/2008 and its amendments).