Reaction mass of (R*,R*)-7-methoxy-3,7-dimethyl-2-octanol and (R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2016 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2016 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Basis for dose level selection: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: MB40RV) following consultation with the Sponsor.
- Route of administration: The dietary route of administration was chosen to simulate the conditions of potential human exposure.
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: Approximately 71 days old; Females: Approximately 85 days old.
- Weight at study initiation: Males: 339-379 g; Females: 241-302 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing one: male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: 02 August 2016 to

Route of administration:

oral: feed

Vehicle:

other: feed

Details on exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved 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 plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C) until the day before use. Formulations were used within 28 h of removal from the freezer.

Details on mating procedure:

- Animals: Toxicity phase and Recovery phase males with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After a minimum of three weeks of treatment.
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.
Animals of the F1 generation received no direct administration of test item; any exposure was in utero or via the milk.

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm (analytical)

Remarks:

Group 1 (control)

Dose / conc.:

1 500 ppm (analytical)

Remarks:

Group 2 (low dose)

Dose / conc.:

3 500 ppm (analytical)

Remarks:

Group 3 (mid dose)

Dose / conc.:

8 000 ppm (analytical)

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: MB40RV) following consultation with the Sponsor. Drastic effects on body weight and food consumption were seen at 15000 ppm at the start of treatment that were considered likely to be related to the palatability of test item. Only partial recovery was observed in females at this dose level after three weeks of treatment, with lower mean body weight than controls (up to - 8%) throughout this period, which might have led to unspecific changes in reproductive parameters in this main study. Therefore, a high dietary concentration of 8000 ppm was chosen, and was expected to elicit initial mean body weight loss or stasis in females and initial low food consumption, as observed in the preliminary toxicity study. The lowest dietary concentration of 1500 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 3500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Abnormal estrus cycle - 11 females; Body weight range extremes - 3 males

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on 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 feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); On the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm). Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity and Recovery phase females. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OTHER:
NEUROBEHAVIOURAL EXAMINATION:
- Time schedule:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.

OPHTHALMOLOGY
- Time schedule:
Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All male Recovery animals
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

Urinalysis
- Time schedule for collection of urine:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery animals
Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis
- Time schedule for examination
At termination: F0 males, All F0 Reproductive phase females
Day 4 of age - F1 offspring, two females per litter (where possible) - no pups were eliminated when litter size dropped below ten/litter
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
# priority given to serum sample
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#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

Estrous Cycle
Wet smears:
Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Dry smears:
Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs (approximately three weeks).

Litter observations:

Clinical observations: Examined at approximately 24 h 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 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Main phase females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Postmortem examinations (offspring):

SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
Method of sacrifice:
- Offspring- selected for thyroid hormone sampling on Day 4 or Day 13 of age: Decapitation
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) 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.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.

Statistics:

See "Any other information on materials and methods incl. tables"

Reproductive indices:

Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100
Gestation Length and Index: Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
Gestation index was calculated for each group as:
- Gestation index (%) = (Number of live litters born / Number pregnant ) x 100

Offspring viability indices:

Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100
Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs considered related to treatment with test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 3500 ppm (3F 78) was killed for welfare reasons on Day 22 of gestation due to general poor clinical condition. Clinical signs included vocalisation, dull eyes, and a firm and prominent area around the vagina. This female was experiencing problems with parturition, and at macroscopic examination was found to have 15 implantations, with one fetus stuck in the cervix and vagina. The placenta was still attached to the uterine wall. At microscopic examination, hemorrhage/inflammatory cell infiltration in the uterus and uterine cervix and inflammatory cell infiltration in the vagina were seen. A relationship to treatment is unlikely as parturition difficulties can be seen in a small percentage of untreated Control animals and no difficulties occurred at 8000 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Bodyweight gain of males receiving test item at 8000 ppm was statistically significantly lower than that of the Control during Week 1 of treatment but was then comparable to the Control thereafter, leading to a 19% decrease of overall bodyweight gain compared to Controls. Overall bodyweight gain was also lower than the Control (-16%) at 3000 ppm whereas overall bodyweight gain of males receiving 1500 ppm was similar to the Control.
- Toxicity and recovery females receiving test item at 3500 or 8000 ppm showed slight bodyweight loss during Week 1, compared to a bodyweight gain amongst Control females or females at 1500 ppm. Females receiving 8000 ppm also showed minor body weight loss during Week 6. Overall body weight gain during Days 1 43 of treatment was statistically significantly lower in females receiving test item at 3500 or 8000 ppm (-53% and -77% than the Control, respectively).
- Overall body weight change during the recovery period was similar to that of the Control for males and females which had received test item at 8000 ppm.
- Overall body weight gain during Days 1 22 of treatment for reproductive phase females (before pairing) was lower than that of the Control in all treated groups (-38%, -44% and -31% at 1500, 3000 and 8000 ppm, respectively). Females at 8000 ppm showed minor mean bodyweight loss during the first week of treatment.
- Overall body weight gain during gestation for reproductive phase females was slightly lower (-8%) than that of the Control for females receiving test item at 1500 ppm, and lower (-13%) than that of the Control for females receiving test item at 3500 or 8000 ppm.
- Overall body weight gain during Days 1-13 of lactation for reproductive phase females was similar to that of the Control for females receiving test item at 1500 ppm, and higher than that of the Control for females receiving test item at 3500 or 8000 ppm (+48% and +33%, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- The food consumption of males receiving test item at 1500, 3500 and 8000 ppm was lower than that of the Control on Day 1 of treatment and generally slightly lower until Day 4/5 and similar to that of the Control thereafter.
- The food consumption of females receiving test item at 8000 ppm was lower than that of the Control on Days 1-3 of treatment and generally similar to that of the Control thereafter. The food consumption of females receiving test item at 3500 ppm was lower than that of the Control on Days 1-5 of treatment and generally similar to that of the Control thereafter. The food consumption of females receiving test item at 1500 ppm was lower than that of the Control on Days 1-2 of treatment and generally similar to that of the Control thereafter.
- Overall food consumption during recovery was similar to that of the Control for males and females which had received test item at 8000 ppm, with a slight increase during the first week of the recovery period.
- Food consumption during Days 1 22 of treatment for reproductive phase females (before pairing) was similar to that of the Control for females receiving test item at 1500 ppm, and lower than that of the Control during the first three days for females receiving test item at 3500 or 8000 ppm.
- Food consumption during gestation for reproductive phase females was generally similar to that of the Control for females receiving test item at 1500 ppm. At 3500 and 8000 ppm, food intake was slightly low on Days 0-3 of gestation, reaching statistical significance on Days 1 and 3.
- Food consumption during Days 1-13 of lactation for reproductive phase females was variable, but generally similar to that of the Control for females receiving test item at 1500, 3500 or 8000 ppm.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic lesions that were considered to be related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology investigations during Week 6 of treatment revealed the following:
- In males: marginally low absolute reticulocyte counts at 8000 ppm, low reticulocyte (%) at 8000 ppm, statistically significantly low mean cell haemoglobin concentration at 8000 ppm, low total and lymphocyte counts at 3500 and 8000 ppm and monocyte counts at 8000 ppm, high platelet counts at 8000 ppm and low activated partial thromboplastin time at 3500 and 8000 ppm.
- In females: marginally high absolute reticulocyte counts at 1500, 3500 or 8000 ppm, marginally high reticulocyte (%) at 1500, 3500 or 8000 ppm, high mean cell haemoglobin concentration at 8000 ppm, and high total and lymphocyte counts at 3500 or 8000 ppm.
- Haematology investigations during recovery revealed no clear differences from Controls for males previously treated with test item at 8000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis after 6 weeks of treatment revealed, when compared to controls:
- In males: high urea and creatinine, and low triglycerides at 8000 ppm
- In females: high urea at 3500 or 8000 ppm, and high cholesterol and calcium in females at 1500, 3500 or 8000 ppm.
- All other biochemical differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
- Blood chemistry investigations during recovery revealed no differences for males and females which had previously received 8000 ppm when compared with that of the Control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis after 6 weeks of treatment revealed, when compared to controls:
- In males: low pH, high specific gravity at 8000 ppm and high chloride
- In females: high glucose, high sodium and high chloride
- All other urinary differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
- Urinalysis investigations during recovery revealed no differences for males and females which had previously received 8000 ppm.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- The sensory reactivity and grip strength observations conducted during Week 6 of treatment revealed no findings which were considered treatment related in either males or females receiving test item at 1500, 3500 or 8000 ppm.
- The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male or female animals at all dose levels.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after 6 weeks of treatment
Treatment related findings: Changes related to treatment with test item were seen in the kidneys.
Kidneys: Hyaline droplet accumulation was seen in all males given 1500 or 3500 or 8000 ppm. Increased severity of tubular basophilia and granular cast(s) were also seen in males given 8000 ppm.
Incidental findings: A mammary adenocarcinoma was seen in one female given 1500 ppm, but was considered to be incidental and not related to treatment as no such change was seen in females of the high dose group and it is seen occasionally in reproductive toxicity studies.
All other histological findings were considered incidental and unrelated to the test item.

Animals killed after 14 days of recovery
Treatment related findings
Kidneys: Minimal hyaline droplet accumulation was seen in one male previously given 8000 ppm for 6 weeks followed by a 14-day recovery period. Based on the reduced incidence and severity of this test item related finding to within the range that is commonly seen as background finding in rat kidneys, it was considered that full recovery had occurred.
Incidental findings: All other histological findings were considered incidental and unrelated to the test item.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
There was no effect of treatment on mean serum T4 concentrations in F0 males or in F1 male and female offspring on Day 13 of age. The mean serum concentration of T4 in Control F0 males was higher than in the Historical Control Data range and no effect of treatment was inferred.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- All females allocated to the study showed regular 4 or 5 day estrus cycles prior to the start of treatment.
- There was no effect of treatment on the estrus cycles of the reproductive phase females. At termination, all reproductive phase females showed diestrous.
- All toxicity and recovery phase females showed estrus before termination. There was no effect of treatment on the stage of the estrus cycle at termination.

Reproductive performance:

no effects observed

Description (incidence and severity):

- There was no effect of treatment on the pre coital interval: the majority of animals mated within 4 days of pairing at the first estrus.
- On this study, there is a definite shift in the number of copulation plugs seen and the sperm count in the vaginal smear in the group 4 animals receiving 8000 ppm. The shift is actually making them superior to Controls, i.e. there are more animals with higher sperm counts and more animals with higher numbers of copulation plugs. The sperm count differences from controls have achieved statistical significance (p<0.01).
- Mating performance was 100% in all groups, while fertility was 80, 90, 100 and 80% in Groups 1, 2, 3 and 4, respectively.
- There was no effect of treatment on gestation length or the gestation index.

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that were considered to be related to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

One female in Group 3 (3F 78) was killed for welfare reasons on Day 22 of gestation. Two females in Group 1 (1F 62 and 1F 65), one female in Group 2 (2F 54), and two females in Group 4 (4F 45 and 4F 48) were killed on Day 25 after mating having failed to litter. There were no signs of any implantations at macroscopic examinations. The following assessment was made based on the 8, 9, 9 and 8 females surviving to termination with a live litter on Day 13 of lactation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on mean body weights of male and female offspring in the 1500, 3500 or 8000 ppm groups on Day 1 of age, or on subsequent bodyweight gain of male and female offspring in the 1500, 3500 or 8000 ppm groups until Day 4 of age. However the bodyweight gain of male and female offspring in the 1500, 3500 or 8000 ppm groups from Day 4 until termination Day 13 of age was marginally lower than that of the Control. This may reflect the slightly higher mean litter size in these groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings in decedent offspring, or offspring at termination on Day 13 of age, that were considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Litter Size, Sex Ratio and Survival Indices: There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio.
Offspring Ano-genital Distance: Ano-genital distance in male and female offspring was unaffected by treatment.

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation Analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500 ppm and 20000 ppm during frozen storage (-10 to -30 °C) for up to 15 days. 500 ppm formulations were confirmed stable for 28 h at ambient storage (15 to 25 °C). 20000 ppm formulations were confirmed stable for 4 days at ambient storage (15 to 25 °C).

The mean concentrations of test item in test formulations analyzed for the study were within 4% of nominal concentrations, confirming accurate formulation.

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 88.5, 204 or 497 mg/kg bw/day respectively at 1500, 3500 or 8000 ppm. 

Mean achieved doses for toxicity and recovery phase females were 90.8, 206 or 469 mg/kg bw/day respectively at 1500, 3500 or 8000 ppm. 

Mean achieved doses for reproductive phase females at 1500, 3500 or 8000 ppm were 91.9, 211 or 497 mg/kg bw/day before pairing, 94.4, 220 or 520 mg/kg bw/day during gestation and 225, 528 or 1241 mg/kg bw/day during Days 1-12 of lactation. The higher achieved intakes during lactation reflect the higher food consumption due to the increased physiological demand on the dams during lactation.

Conclusions:

Based on the results of this study it is concluded that the NOAEL of test item for systemic toxicity was 8000 ppm (mean achieved doses of 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females, 520 mg/kg bw/day for females during gestation and 1241 mg/kg bw/day for females during lactation). The NOAEL of the test item for reproductive/developmental effects was concluded to be 8000 ppm.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 3500 and 8500 ppm.

 

Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. The F1 generation was killed on Day 4 or Day 13 of age, but received no direct administration of test item; any exposure was in utero or via the milk. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, nipple counts (males only), ano-genital distance and macropathology for all offspring were also assessed. 

 

The mean concentrations of test item in test formulations analyzed for the study were within 4% of nominal concentrations, confirming accurate formulation. Achieved dose, generally maintained the intervals between dietary concentrations. During lactation, achieved doses were higher, reflecting the increased physiological demand on the dams.

There was no effect of treatment on mean serum T4 concentrations in F0 males or in F1 male and female offspring on Day 13 of age.

Dietary administration of test item at levels of 1500, 3500 and 8000 ppm was generally well tolerated. There was one death on Day 22 of gestation at 3500 ppm which was considered not to be related to treatment. There were no adverse effects attributed to treatment on clinical condition, sensory reactivity, grip strength, motor activity, ophthalmic examination, hematology, blood chemistry, urinalysis and macroscopic pathology. In addition, the reproductive endpoints assessed which were unaffected by treatment included estrus cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival, sex ratio or external development. 

Bodyweight gain of males receiving test item at 8000 ppm was statistically significantly lower than that of the Control during Week 1 of treatment but was then comparable to the Control thereafter, leading to a 19% decrease of overall bodyweight gain compared to Controls. Overall bodyweight gain was also lower than the Control (-16%) at 3000 ppm whereas overall bodyweight gain of males receiving 1500 ppm was similar to the Control. Toxicity and recovery females receiving test item at 3500 or 8000 ppm showed slight bodyweight loss during Week 1, compared to a bodyweight gain amongst Control females or females at 1500 ppm. Overall body weight gain during Days 1‑43 of treatment was statistically significantly lower in females receiving test item at 3500 or 8000 ppm. Overall body weight change during the recovery period was similar to that of the Control for males and females which had received test item at 8000 ppm. Overall body weight gain during Days 1‑22 of treatment for reproductive phase females (before pairing) was lower than that of the Control in all treated groups. Females at 8000 ppm showed minor mean bodyweight loss during the first week of treatment. Overall body weight gain during gestation for reproductive phase females was slightly lower than that of the Control for females receiving test item at 1500 ppm, and lower than that of the Control for females receiving test item at 3500 or 8000 ppm. Overall body weight gain during Days 1-13 of lactation for reproductive phase females was similar to that of the Control for females receiving test item at 1500 ppm, and higher than that of the Control for females receiving test item at 3500 or 8000 ppm.

Food consumption of males receiving 1500, 3500 and 8000 ppm was low on day 1 of treatment and slightly low until Day 4/5. Food consumption of treated females was also low during the first few days of treatment. Overall food consumption during recovery was unaffected by previous treatment. At 3500 and 8000 ppm, mean food consumption was slightly low during Days 0-3 of gestation. There was no conclusive effect of treatment on food intake during lactation.

After 6 weeks of treatment, the mean absolute and adjusted liver weights for males and females receiving 1500, 3500 or 8000 ppm were marginally high. After 2 weeks recovery, the mean absolute and adjusted liver weights for males and females that previously received 8000 ppm were similar to Control. On Day 13 of lactation, the adjusted ovary weights were lower than Control at 8000 ppm, and the adjusted mean uterus and cervix were lower than Control at 1500, 3500 or 8000 ppm. Histopathological examination of these tissues showed no abnormalities and reproductive performance and litter size of these females was unaffected by treatment, therefore no effect of treatment is inferred.

Dietary administration of test item to rats for 6 weeks resulted in test item related findings in the kidneys of all treated males. Hyaline droplet accumulation can be seen as a background change in rat kidneys, but the incidence and severity of this change in males and the association with an increased severity of tubular basophilia and the presence of granular casts indicate that this change is related to treatment. Hyaline droplet accumulation is rodent-specific and has no toxicological significance in humans. Following a 14-day recovery period, full recovery from test item related findings seen in the kidneys was considered to have occurred.

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of the test item for systemic toxicity was 8000 ppm (mean achieved doses of 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females, 520 mg/kg bw/day for females during gestation and 1241 mg/kg bw/day for females during lactation). The NOAEL of the test item for reproductive/developmental effects was 8000 ppm.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

An oral diet repeated dose toxicity study (combined with a reproduction/developmental toxicity screening test) was conducted with the registered substance according to Guideline OECD 422 and in compliance with GLP. The NOAEL for systemic  toxicity was 8000 ppm, the highest dose tested, corresponding to 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females and 497 mg/kg/day for reproductive phase females during pre-mating period.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

488 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Recent GLP study conducted according to OECD Guideline No 422 without any deviation (Klimisch score = 1).

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

No adverse toxic effects were observed in a repeated dose toxicity study until the highest dose tested, corresponding to 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females and 497 mg/kg/day for reproductive phase females during pre-mating period, which is above the classification threshold of 300 mg/kg bw/day for a sub-acute study.

Therefore the registered substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No 1272 /2008.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-57-0

Molecular formula:

C11H24O2

IUPAC Name:

(R*,R*)-7-methoxy-3,7-dimethyl-2-octanol

Constituent 2

Reference substance name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

Cas Number:

87605-61-6

Molecular formula:

C11H24O2

IUPAC Name:

(R*,S*)-7-methoxy-3,7-dimethyl-2-octanol

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 179259
Purity: 99.6% (sum of the two main constituents)
Name of test material (as cited in study report): 7-METHOXY-3,7-DIMETHYLOCTAN-2-OL MULTICONSTITUENT
Physical state: colourless - slightly yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 09 June 2018

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: Approximately 71 days old; Females: Approximately 85 days old.
- Weight at study initiation: Males: 339-379 g; Females: 241-302 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing one: male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: 02 August 2016 to

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

other: feed

Details on oral exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved 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 plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C) until the day before use. Formulations were used within 28 h of removal from the freezer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

Group 2 (low dose)

Doses / concentrations 3

Dose / conc.:

3 500 ppm

Remarks:

Group 3 (mid dose)

Doses / concentrations 4

Dose / conc.:

8 000 ppm

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: MB40RV) following consultation with the Sponsor. Drastic effects on body weight and food consumption were seen at 15000 ppm at the start of treatment that were considered likely to be related to the palatability of test item. Only partial recovery was observed in females at this dose level after three weeks of treatment, with lower mean body weight than controls (up to - 8%) throughout this period, which might have led to unspecific changes in reproductive parameters in this main study. Therefore, a high dietary concentration of 8000 ppm was chosen, and was expected to elicit initial mean body weight loss or stasis in females and initial low food consumption, as observed in the preliminary toxicity study. The lowest dietary concentration of 1500 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 3500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
- Post-exposure recovery period in satellite groups: 14 days
- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Abnormal estrus cycle - 11 females; Body weight range extremes - 3 males

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on 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 feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); On the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm). Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity and Recovery phase females. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All male Recovery animals
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery animals
Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Other examinations:

Thyroid Hormone Analysis:
At termination: F0 males, All F0 Reproductive phase females

Statistics:

See "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs considered related to treatment with test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 3500 ppm (3F 78) was killed for welfare reasons on Day 22 of gestation due to general poor clinical condition. Clinical signs included vocalisation, dull eyes, and a firm and prominent area around the vagina. This female was experiencing problems with parturition, and at macroscopic examination was found to have 15 implantations, with one fetus stuck in the cervix and vagina. The placenta was still attached to the uterine wall. At microscopic examination, hemorrhage/inflammatory cell infiltration in the uterus and uterine cervix and inflammatory cell infiltration in the vagina were seen. A relationship to treatment is unlikely as parturition difficulties can be seen in a small percentage of untreated Control animals and no difficulties occurred at 8000 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Bodyweight gain of males receiving test item at 8000 ppm was statistically significantly lower than that of the Control during Week 1 of treatment but was then comparable to the Control thereafter, leading to a 19% decrease of overall bodyweight gain compared to Controls. Overall bodyweight gain was also lower than the Control (-16%) at 3000 ppm whereas overall bodyweight gain of males receiving 1500 ppm was similar to the Control.
- Toxicity and recovery females receiving test item at 3500 or 8000 ppm showed slight bodyweight loss during Week 1, compared to a bodyweight gain amongst Control females or females at 1500 ppm.
Females receiving 8000 ppm also showed minor body weight loss during Week 6. Overall body weight gain during Days 1 43 of treatment was statistically significantly lower in females receiving test item at 3500 or 8000 ppm (-53% and -77% than the Control, respectively).
- Overall body weight change during the recovery period was similar to that of the Control for males and females which had received test item at 8000 ppm.
- Overall body weight gain during Days 1 22 of treatment for reproductive phase females (before pairing) was lower than that of the Control in all treated groups (-38%, -44% and -31% at 1500, 3000 and 8000 ppm, respectively). Females at 8000 ppm showed minor mean bodyweight loss during the first week of treatment.
- Overall body weight gain during gestation for reproductive phase females was slightly lower (-8%) than that of the Control for females receiving test item at 1500 ppm, and lower (-13%) than that of the Control for females receiving test item at 3500 or 8000 ppm.
- Overall body weight gain during Days 1-13 of lactation for reproductive phase females was similar to that of the Control for females receiving test item at 1500 ppm, and higher than that of the Control for females receiving test item at 3500 or 8000 ppm (+48% and +33%, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- The food consumption of males receiving test item at 1500, 3500 and 8000 ppm was lower than that of the Control on Day 1 of treatment and generally slightly lower until Day 4/5 and similar to that of the Control thereafter.
- The food consumption of females receiving test item at 8000 ppm was lower than that of the Control on Days 1-3 of treatment and generally similar to that of the Control thereafter. The food consumption of females receiving test item at 3500 ppm was lower than that of the Control on Days 1-5 of treatment and generally similar to that of the Control thereafter. The food consumption of females receiving test item at 1500 ppm was lower than that of the Control on Days 1-2 of treatment and generally similar to that of the Control thereafter.
- Overall food consumption during recovery was similar to that of the Control for males and females which had received test item at 8000 ppm, with a slight increase during the first week of the recovery period.
- Food consumption during Days 1 22 of treatment for reproductive phase females (before pairing) was similar to that of the Control for females receiving test item at 1500 ppm, and lower than that of the Control during the first three days for females receiving test item at 3500 or 8000 ppm.
- Food consumption during gestation for reproductive phase females was generally similar to that of the Control for females receiving test item at 1500 ppm. At 3500 and 8000 ppm, food intake was slightly low on Days 0-3 of gestation, reaching statistical significance on Days 1 and 3.
- Food consumption during Days 1-13 of lactation for reproductive phase females was variable, but generally similar to that of the Control for females receiving test item at 1500, 3500 or 8000 ppm.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic lesions that were considered to be related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology investigations during Week 6 of treatment revealed the following:
- In males: marginally low absolute reticulocyte counts at 8000 ppm, low reticulocyte (%) at 8000 ppm, statistically significantly low mean cell haemoglobin concentration at 8000 ppm, low total and lymphocyte counts at 3500 and 8000 ppm and monocyte counts at 8000 ppm, high platelet counts at 8000 ppm and low activated partial thromboplastin time at 3500 and 8000 ppm.
- In females: marginally high absolute reticulocyte counts at 1500, 3500 or 8000 ppm, marginally high reticulocyte (%) at 1500, 3500 or 8000 ppm, high mean cell haemoglobin concentration at 8000 ppm, and high total and lymphocyte counts at 3500 or 8000 ppm.
- Haematology investigations during recovery revealed no clear differences from Controls for males previously treated with test item at 8000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis after 6 weeks of treatment revealed, when compared to controls:
- In males: high urea and creatinine, and low triglycerides at 8000 ppm
- In females: high urea at 3500 or 8000 ppm, and high cholesterol and calcium in females at 1500, 3500 or 8000 ppm.
- All other biochemical differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
- Blood chemistry investigations during recovery revealed no differences for males and females which had previously received 8000 ppm when compared with that of the Control.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis after 6 weeks of treatment revealed, when compared to controls:
- In males: low pH, high specific gravity at 8000 ppm and high chloride
- In females: high glucose, high sodium and high chloride
- All other urinary differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
- Urinalysis investigations during recovery revealed no differences for males and females which had previously received 8000 ppm.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- The sensory reactivity and grip strength observations conducted during Week 6 of treatment revealed no findings which were considered treatment related in either males or females receiving test item at 1500, 3500 or 8000 ppm.
- The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male or female animals at all dose levels.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- After 6 weeks of treatment, the mean absolute and adjusted liver weights for males and females receiving 1500, 3500 or 8000 ppm were marginally high, especially at the high dose (absolute weights: +26% and +27% than Control in males and females, respectively). After 2 weeks recovery, the absolute and mean adjusted liver weights for males and females that previously received 8000 ppm were similar to Control.
- On Day 13 of lactation, the adjusted ovary weights were statistically significantly lower than Control at 8000 ppm, and the adjusted mean uterus and cervix were lower than Control at 1500, 3500 or 8000 ppm. Histopathological examination of these tissues showed no abnormalities, therefore no effect of treatment was inferred.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Animals Killed Day 13 of Lactation
- The macroscopic examination performed after day 13 of lactation revealed no test item related lesions.
- The incidence and distribution of all findings were considered to be incidental and unrelated to treatment.

Animals Killed After 6 Weeks of Treatment
- The macroscopic examination performed after 6 weeks of treatment revealed no test item related lesions.
- The incidence and distribution of all findings were considered to be incidental and unrelated to treatment.

Animals Killed After 14 Days of Recovery
- The macroscopic examination performed after 14 days of recovery revealed no test item related lesions.
- The incidence and distribution of all findings were considered to be incidental and unrelated to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after 6 weeks of treatment
Treatment related findings: Changes related to treatment with test item were seen in the kidneys.
Kidneys: Hyaline droplet accumulation was seen in all males given 1500 or 3500 or 8000 ppm. Increased severity of tubular basophilia and granular cast(s) were also seen in males given 8000 ppm.
Incidental findings: A mammary adenocarcinoma was seen in one female given 1500 ppm, but was considered to be incidental and not related to treatment as no such change was seen in females of the high dose group and it is seen occasionally in reproductive toxicity studies.
All other histological findings were considered incidental and unrelated to the test item.

Animals killed after 14 days of recovery
Treatment related findings
Kidneys: Minimal hyaline droplet accumulation was seen in one male previously given 8000 ppm for 6 weeks followed by a 14-day recovery period. Based on the reduced incidence and severity of this test item related finding to within the range that is commonly seen as background finding in rat kidneys, it was considered that full recovery had occurred.
Incidental findings: All other histological findings were considered incidental and unrelated to the test item.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
There was no effect of treatment on mean serum T4 concentrations in F0 males. The mean serum concentration of T4 in Control F0 males was higher than in the Historical Control Data range and no effect of treatment was inferred.

Effect levels

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Formulation Analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500 ppm and 20000 ppm during frozen storage (-10 to -30 °C) for up to 15 days. 500 ppm formulations were confirmed stable for 28 h at ambient storage (15 to 25 °C). 20000 ppm formulations were confirmed stable for 4 days at ambient storage (15 to 25 °C).

The mean concentrations of test item in test formulations analyzed for the study were within 4% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 88.5, 204 or 497 mg/kg bw/day respectively at 1500, 3500 or 8000 ppm.

Mean achieved doses for toxicity and recovery phase females were 90.8, 206 or 469 mg/kg bw/day respectively at 1500, 3500 or 8000 ppm.

Mean achieved doses for reproductive phase females at 1500, 3500 or 8000 ppm were 91.9, 211 or 497 mg/kg bw/day before pairing, 94.4, 220 or 520 mg/kg bw/day during gestation and 225, 528 or 1241 mg/kg bw/day during Days 1-12 of lactation. The higher achieved intakes during lactation reflect the higher food consumption due to the increased physiological demand on the dams during lactation.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the NOAEL of the test item for systemic toxicity was 8000 ppm (mean achieved doses of 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females, 520 mg/kg bw/day for females during gestation and 1241 mg/kg bw/day for females during lactation).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 3500 and 8000 ppm.

 

Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

The mean concentrations of test item in test formulations analyzed for the study were within 4% of nominal concentrations, confirming accurate formulation. Achieved dose, generally maintained the intervals between dietary concentrations. During lactation, achieved doses were higher, reflecting the increased physiological demand on the dams.

 

There was no effect of treatment on mean serum T4 concentrations in F0 males.

 

Dietary administration of test item at levels of 1500, 3500 and 8000 ppm was generally well tolerated. There was one death on Day 22 of gestation at 3500 ppm which was considered not to be related to treatment. There were no adverse effects attributed to treatment on clinical condition, sensory reactivity, grip strength, motor activity, ophthalmic examination, hematology, blood chemistry, urinalysis and macroscopic pathology.

 

Bodyweight gain of males receiving test item at 8000 ppm was statistically significantly lower than that of the Control during Week 1 of treatment but was then comparable to the Control thereafter, leading to a 19% decrease of overall bodyweight gain compared to Controls. Overall bodyweight gain was also lower than the Control (-16%) at 3000 ppm whereas overall bodyweight gain of males receiving 1500 ppm was similar to the Control. Toxicity and recovery females receiving test item at 3500 or 8000 ppm showed slight bodyweight loss during Week 1, compared to a bodyweight gain amongst Control females or females at 1500 ppm. Overall body weight gain during Days 1‑43 of treatment was statistically significantly lower in females receiving test item at 3500 or 8000 ppm. Overall body weight change during the recovery period was similar to that of the Control for males and females which had received test item at 8000 ppm. Overall body weight gain during Days 1‑22 of treatment for reproductive phase females (before pairing) was lower than that of the Control in all treated groups. Females at 8000 ppm showed minor mean bodyweight loss during the first week of treatment. Overall body weight gain during gestation for reproductive phase females was slightly lower than that of the Control for females receiving test item at 1500 ppm, and lower than that of the Control for females receiving test item at 3500 or 8000 ppm. Overall body weight gain during Days 1-13 of lactation for reproductive phase females was similar to that of the Control for females receiving test item at 1500 ppm, and higher than that of the Control for females receiving test item at 3500 or 8000 ppm.

 

Food consumption of males receiving 1500, 3500 and 8000 ppm was low on day 1 of treatment and slightly low until Day 4/5. Food consumption of treated females was also low during the first few days of treatment. Overall food consumption during recovery was unaffected by previous treatment. At 3500 and 8000 ppm, mean food consumption was slightly low during Days 0-3 of gestation. There was no conclusive effect of treatment on food intake during lactation.

 

After 6 weeks of treatment, the mean absolute and adjusted liver weights for males and females receiving 1500, 3500 or 8000 ppm were marginally high. After 2 weeks recovery, the mean absolute and adjusted liver weights for males and females that previously received 8000 ppm were similar to Control. On Day 13 of lactation, the adjusted ovary weights were lower than Control at 8000 ppm, and the adjusted mean uterus and cervix were lower than Control at 1500, 3500 or 8000 ppm. Histopathological examination of these tissues showed no abnormalities and reproductive performance and litter size of these females was unaffected by treatment, therefore no effect of treatment is inferred.

 

Dietary administration of test item to rats for 6 weeks resulted in test item related findings in the kidneys of all treated males. Hyaline droplet accumulation can be seen as a background change in rat kidneys, but the incidence and severity of this change in males and the association with an increased severity of tubular basophilia and the presence of granular casts indicate that this change is related to treatment. Hyaline droplet accumulation is rodent-specific and has no toxicological significance in humans. Following a 14-day recovery period, full recovery from test item related findings seen in the kidneys was considered to have occurred.

 

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) of the test item for systemic toxicity was 8000 ppm (mean achieved doses of 497 mg/kg bw/day for males, 469 mg/kg bw/day for toxicity phase females, 520 mg/kg bw/day for females during gestation and 1241 mg/kg bw/day for females during lactation).