2-(3,3-dimethylcyclohex-1-enyl)-2,5,5-trimethyl-1,3-dioxane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 August 2019 to 12 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 422 without any deviation

Cross-reference

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:

From 30 August 2019 to 12 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 422 without any deviation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2021-02-12

Limit test:

no

Species:

rat

Strain:

Crj: 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) rat 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 nulliparous and non-pregnant: yes
- Age at study initiation: Males 71 to 77 days old / Females 85 to 92 days old
- Weight at study initiation: Males 326 to 406 g / Females 224 to 296 g
- Fasting period before study: no
- 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
Pre-pairing, toxicity phase females and recovery animals: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: one female
Lactation: one female + litter
- Diet (e.g. ad libitum): Non-restricted SDS VRF1 Certified powdered diet
- Water (e.g. ad libitum): Non-restricted. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 8 days (Males) / 22 days (Females).
- Environmental enrichment: A soft white untreated wood block and a plastic shelter were provided to each cage throughout the study (except during lactation) and replaced
when necessary or at the same time as the cages. In addition, approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation and changed at the same frequency as cage bedding.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-09-04 To: 2019-12-11

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): SDS VRF1 Certified powdered diet
- Storage temperature of food: At ambient temperature (15 to 25°C) for up to 24 days.
- Method of preparation: The test item was incorporated into the plain 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 item was weighed into a suitable container. An amount of diet that approximately equaled the weight of test item was added and the mixture stirred together. A further amount of diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder, after which it was made up to the final weight of the premix with diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

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.
In this study the homogeneity and stability of MADERAL ZA1076 in the diet matrix was demonstrated for up to 24 days when stored at ambient (15 to 25°C) or frozen temperature (-10 to -30°C).
- Achieved concentration:
Samples of each formulation prepared for administration in Week 1 of treatment and on Day 12 of lactation were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks.
Recovery phase animals were treated for six weeks followed by a 28-day recovery period.
Reproductive phase females were treated for two weeks before pairing, throughout pairing and gestation and until Day 12 of lactation.

Frequency of treatment:

Continuous

Dose / conc.:

1 000 ppm

Remarks:

54.9 mg/kg/day for males and 65.4 mg/kg/day for females

Dose / conc.:

3 000 ppm

Remarks:

169 mg/kg/day for males and 214 mg/kg/day for females

Dose / conc.:

10 000 ppm

Remarks:

551 mg/kg/day for males and 625 mg/kg/day for females

No. of animals per sex per dose:

- Reproductive phase: 10 Females
- Toxicity phase: 10 Males / 5 Females
- Recovery: 5 Males and 5 Females (control and high dose groups)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations selected for this OECD 422 study based on the results of a 14-day preliminary toxicity study.
In the preliminary toxicity study, dietary concentrations of 1500, 5000 and 15000 ppm were investigated. Males receiving 15000 showed low body weight gain between Days 1 and 4, but good weight gain thereafter. After 14 Days of feeding of treated diet the group mean body weight was similar for males treated with 1500, 5000 or 15000 ppm Maderal ZA1076. For females fed with diet containing 15000 ppm group mean body weight loss was apparent up until Day 4 of treatment, a general improvement in body weight performance was then seen in females for the remainder of the treatment period. Overall females had a small group mean body weight loss by Day 15 of treatment.
A reduction in food intake was observed from Day 1-4 of treatment in both males and females fed treated diet containing 15000 ppm. An improvement in food consumption was observed throughout the remainder of the treatment period, such that food consumption was similar throughout the groups by the end of the first week of treatment. There were no test item related macroscopic findings. Both males receiving 15000 ppm and females receiving 5000 or 15000 ppm had increased liver weights. All other organ weights were similar throughout the groups.
Based on the results of this preliminary toxicity study, a high dietary level of 10000 ppm an intermediate level of 3000ppm, and a low level of 1000 ppm were selected for the Main study.
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: used to assess reversibility, persistence or delayed occurrence of systemic effects
- Post-exposure recovery period in satellite groups: 28 days

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, on Days 0, 7, 14 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.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization (not reported).
Before feeding of the treated diets on the day that treatment commenced and weekly thereafter (including recovery phase) and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization (not reported).
Before feeding of the treated diets on the day that treatment commenced and weekly before pairing.
Days 0, 7, 14, and 20 after mating and Days 1, 4, 7, and 13 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Days 22 to 28). Food consumption was recorded continuously for Toxicity phase females and Recovery phase animals.
For Reproductive phase females after mating food consumption was recorded to match the body weight schedule: Days 0-6, 7-13, 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination, or on Day 13 of lactation (lactating females)
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- How many animals: The five lowest numbered surviving Toxicity phase males per group. All Toxicity phase females. The first five lactating females with a litter per group (Reproductive phase). All Recovery phase males and females.
- Parameters checked in table 7.6.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination, or on Day 13 of lactation (lactating females).
- Animals fasted: No
- How many animals: The five lowest numbered surviving Toxicity phase males per group. All Toxicity phase females. The first five lactating females with a litter per group (Reproductive phase). All Recovery phase males and females.
- Parameters checked in table 7.6.1/1 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Parameters examined: Thyroid stimulating hormone (TSH) and Thyroxine (T4) from sublingual vein blood sample site from an isoflurane anesthesia.
- Time of blood sample collection and how many animals:
At termination: All surviving F0 males (Toxicity and Recovery phase) and Toxicity and Recovery phase females. All surviving F0 Reproductive phase females.
Day 4 of age: up to two females per litter (where possible)
Day 13 of age: two males and two females per litter (where possible)
- Animals fasted: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: on the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 5 of treatment and on the first five lactating main study females in each group at Days 7-9 of lactation.
- Battery of functions tested: sensory activity and grip strength (approach response, pinna reflex, auditory startle reflex and tail pinch response) / motor activity

IMMUNOLOGY: No

PARTURITION OBSERVATIONS: Yes
- 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):

- Dry smears:
Reproductive phase females: For 15 days before pairing using cotton swabs.
- Wet smears:
Using pipette lavage.
After pairing until mating (Reproductive phase females).
For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Sperm parameters (parental animals):

Parameters examined in the five lowest numbered surviving toxicity phase males in each group: testis weight, epididymis weight, 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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
Clinical observations were made 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.
Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.

The following parameters were examined in F1 offspring:
number and sex of pups (sex ratio on days 1, 4, 7 and 13 of age), stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) for all offspring at Day 1, pup weight on the days 1, 4, 7 and 13 of age, presence of nipples/areolae in male pups at day 13 of age.

GROSS EXAMINATION OF DEAD PUPS:
yes, 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. Any abnormal tissues were retained.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
1/ On day 4 of age: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation):
• one for T4 (serum)#
• one for TSH (serum)
# Priority given to T4 sample.
No offspring were allocated to these procedures on Day 4 of age if:
• the resultant live litter size would fall below ten offspring
• the resultant number of female pups would fall below three offspring
• If only four female offspring were available within a litter but the overall litter size was >ten, one female was selected with priority given to the T4 sample.

2/ On 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 (serum); where possible one male and one female
# Priority given to T4 sample.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 6 weeks of treatment
- Maternal animals: All surviving animals on Day 13 of lactation

GROSS PATHOLOGY: Yes (see table 7.6.1/2-3-4)

HISTOPATHOLOGY: Yes (see table 7.6.1/2-3-4)

Postmortem examinations (offspring):

SACRIFICE
- Selected F1 offspring were sampled and killed on Day 4 (not analyzed) or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age.

GROSS NECROPSY
- 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. Any abnormal tissues were retained.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non significant, are presented on the relevant tables. For some parameters, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption during the treatment phase and the before pairing phase for Reproductive phase females were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during treatment, gestation and lactation
Hematology
Blood chemistry
Estrous cycles during treatment
Pre-coital interval
Gestation length
Stage of estrous cycle at termination (Toxicity phase)
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index,
live birth index and viability index), for before blood sampling study periods
Ano-genital distance, adjusted for Day 1 pup body weight
Organ weights, absolute and adjusted for terminal body weight

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

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

The sequence of statistical tests used for each parameters were detailed below.

Reproductive indices:

Percentage mating (%) = Number of animals mating x 100 / Animals paired

Conception rate (%) = Number of animals achieving pregnancy x 100 / Animals mated

Fertility index (%) = Number of animals achieving pregnancy x 100 / Animals paired

Gestation index (%) = Number of live litters born x 100 / Number pregnant

Offspring viability indices:

Post-implantation survival index (%) = Total number of offspring born x 100 / Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = Number of live offspring on Day 1 after littering x 100 / Total number of offspring born

Viability index (%) = Number of live offspring on Day 4 (before blood sampling) x 100 / Number live offspring on Day 1 after littering

Lactation index (%) = Number of live offspring on Day 13 after littering x 100 / Number of live offspring on Day 4 (after blood sampling)

Group mean values were calculated from individual litter values

Clinical signs:

no effects observed

Description (incidence and severity):

There were no findings during the detailed physical examination and arena investigations associated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in males fed 3000 or 10000 ppm MADERAL ZA1076 (80% of the Controls for both groups) during the first week of treatment: and slightly reduced in all treated groups from Day 22-29. Toxicity phase females fed diet containing 10000 ppm showed minor weight loss in first week of treatment, weight gain remained reduced during Days 15-43 of treatment. Reproductive females treated with 10000 ppm prior to pairing had reduced body weight gain prior to pairing and during gestation (Days 0-7, 7-14, and 14-20), when compared with the Controls. At the beginning of the recovery period, males and females previously treated with 10000 ppm had a reduced group mean body weight when compared with the Control group. Body weight gain for males and females previously treated with 10000 ppm was increased when compared with the Controls; hence displaying initial signs of recovery from any treatment related body weight effect. By the end of the four-week recovery period, the mean body weight of the males previously treated with 10000 ppm was similar to the mean body weight of the Control males, and the mean body weight of the females previously treated with 10000 ppm was slightly reduced when compared with the mean body weight of the Control females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food intake was reduced in all groups receiving treated diet during the first week of treatment and remained reduced in toxicity phase females receiving 1000 or 10000 ppm throughout the remainder of the treatment period. During treatment and prior to pairing females fed 10000 ppm had a reduced group mean food intake when compared with the controls. These females continued to have reduced food consumption throughout gestation and lactation, respectively. Food consumption for males and females previously treated with 10000 ppm was similar to the controls for the last three weeks of recovery, food consumption for previously treated females during the first week of recovery was reduced when compared with the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological examination in Week 7 did identify differences from the control that were likely to be attributable to treatment; therefore further investigation during recovery was instigated.
In Week 7 for both sexes treated with 10000 ppm, the white blood cell, neutrophil, lymphocyte, eosinophil, monocyte count and APTT were all reduced when compared with the control group. Large unstained cells were increased in all treated groups of males. The haematocrit and mean cell hemoglobin in females during Week 7 were reduced; this effect was not seen in the males. Further investigation to determine any recovery from these effects was performed on these parameters.
By the end of recovery for males and females previously treated with 10000 ppm, white blood cell and lymphocyte counts were all increased when compared with the Controls. Large unstained cells remained slightly increased in males after four weeks of recovery, neutrophil count was increased and APTT remained reduced in females previously treated with 10000 ppm when compared with Controls.
On Day 13 of lactation similar parameters were affected by treatment in females receiving 1000 or 10000 ppm; white blood cell, neutrophil, lymphocyte and eosinophil count and APTT were all reduced. However, females treated with 3000 ppm generally had marginally increased or similar values to the controls for these parameters.
All other differences from control were minor or did not show relationship to dose or confined to one sex, and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination in Week 7, revealed increased plasma urea concentrations, increased plasma cholesterol and triglyceride concentrations in both sexes receiving 10000 ppm. Increased plasma glucose concentrations were seen in all treated groups of males. Reduced phosphorus concentrations were seen in both sexes receiving 3000 or 10000 ppm. Slightly increased total protein concentration, increased albumin and globulin were seen in toxicity phase animals receiving 3000 or 10000 ppm. Chloride concentrations in males treated with 10000 ppm was also reduced. Further investigation to determine any recovery from these effects was performed on these parameters. Plasma urea, cholesterol and triglyceride concentrations were comparable with the Control group after four weeks of recovery in males and females previously treated with 10000 ppm. Reduced phosphorus concentrations remained in both sexes after four weeks of recovery, but chloride concentrations in males following four weeks of recovery were similar to the plasma chloride concentrations in Control males, and protein, albumin and globulin concentrations were generally similar to Controls.
On Day 13 of lactation, plasma urea concentrations, plasma glucose concentrations, plasma cholesterol and triglyceride concentrations displayed the same treatment related effects as seen in the toxicity animals. However, no major differences between treated animals and the controls were seen in phosphorus, total protein concentrations and albumin or globulin concentrations. Alkaline phosphatase and alanine transferase activities were low in all treated groups; attaining statistical significance in all treated groups for alanine transferase and females treated with 10000 ppm for alkaline phosphatase. However, a reduction in enzyme activity is generally considered non adverse and of no toxicological importance. Bile acid concentrations were increased for females receiving 10000 ppm; however, there was considerable inter-group variation and the difference did not achieve statistical significance. Sodium and chloride concentrations were slightly high at 3000 or 10000 ppm.

Endocrine findings:

no effects observed

Description (incidence and severity):

The T4 concentrations in the offspring were comparable with endogenous levels in all treatment groups, and the T4 concentrations in the adult males were not significantly reduced; therefore, no effect of treatment is inferred.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Sensory Reactivity Observations and Grip Strength:
There were no changes in sensory reactivity or grip strength attributable to treatment.
When compared with the control, hindlimb grip strength was low in females on Day 7-9 of lactation receiving 1000 ppm. However, as there is no dose relationship this is likely attributed to natural variation and is therefore not an effect of treatment; hence, no sensory reactivity or grip strength investigations were performed during recovery.
- Motor activity:
Group mean activity scores for all groups of treated males during Week 5 are low when compared with the controls, with several 18 and 36 minute high and low beam interval scores achieving statistical significance in the males. All but one of these scores are within the HCD range; so no effect of treatment is inferred. There was no effect of treatment on the activity of toxicity phase females at 10000 ppm. Females receiving 3000 or 10000 ppm on Day 7-9 of lactation have decreased high and low mean beam scores; all of which are within the HCD range. Due to these scores being within the HCD range, there was not considered to be a treatment related effect and therefore motor activity investigations were not performed during recovery.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1/ Animals Killed After 6 Weeks of Treatment
Changes related to treatment with MADERAL ZA1076 at 10000 ppm were seen in the liver, thyroids and adrenals of males and females and the kidneys of males.
- Liver: Minimal centrilobular hepatocellular hypertrophy was seen in all animals treated with 10000 ppm.
- Thyroids: Minimal follicular cell hypertrophy/hyperplasia was seen at a higher incidence in males and females treated with 10000 ppm than in the respective control animals.
- Adrenals: Minimal diffuse hypertrophy of the zona glomerulosa was seen in the majority of animals treated with 10000 ppm.
- Kidneys: A minimal to moderate accumulation of hyaline droplets in the cortical tubular epithelium was seen in all males treated with 10000 ppm. The kidneys of animals treated with 1000 or 3000 ppm were only examined if macroscopic changes were seen at necropsy. At microscopy, minimal and slight accumulation of hyaline droplets was seen in one male of the groups treated with 1000 or 3000 ppm respectively. Minimal or slight basophilia of cortical tubules was seen in the majority of males treated with 10000 ppm and at the minimal degree in one control male.

2/ Animals Killed After 4 Weeks of Recovery
Kidneys: A minimal accumulation of hyaline droplets in the cortical tubular epithelium was seen in one male control. Minimal or slight basophilia of cortical tubules was seen in the majority of males that were previously treated with 10000 ppm and at the minimal degree in one control male.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Prior to treatment all females had a regular estrus cycle of 4 to 5 days. During treatment, all control females had a regular estrus cycle. But for females treated with MADERAL ZA1076, all females did not cycle regularly. For females receiving 1000 ppm two females had an irregular cycle and four females showed acyclicity. For females receiving 3000 ppm two females exhibited acyclicity, and for females receiving 10000 ppm one female had an irregular cycle and two females exhibited acyclicity.
The increase in acyclicity and irregular cycles in treated females did not affect mating performance and fertility, as all females displayed a 100% conception rate.
There was no effect of treatment on estrous cycles at termination; all reproductive phase females were in diestrus.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

All females displayed a 100% conception rate.
The length of pre-coital interval was slightly extended in females receiving 1000, 3000 or 10000 ppm. Gestation length and index were unaffected by treatment with MADERAL ZA1076.

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity Males

Effect level:

>= 551 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity Females

Effect level:

>= 625 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Females during gestation

Effect level:

>= 609 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Females during lactation

Effect level:

>= 1 332 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no in-life clinical signs reported in offspring which were considered test-item related, following parental treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Litter size was unaffected by treatment with MADERAL ZA1076.
Post implantation survival index (%), live birth index (%) and viability index (%) Day 4 were all considered to be unaffected by treatment. The sex ratio of males to females (%M) was slightly high in litters from mothers treated with 10000 ppm; however there was considerable inter-group variation and therefore this is not considered to be an effect of treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

From Day 4 of age, generally the offspring born to mothers treated with 10000 ppm had slightly reduced body weights than offspring born to control mothers. On Day 13 of age, female offspring from treated mothers receiving 10000 ppm had lower body weights when compared with the offspring from control mothers (85% of the controls). The overall body weight gain (Day 1-13) for male and female offspring from mothers receiving 10000 ppm is markedly low (81% of the Controls for both male and female offspring).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect of treatment on ano-genital distance in male or female offspring, following parental treatment at 1000, 3000 or 10000 ppm; minor differences in female offspring were considered to be of no biological significance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Male offspring did not develop nipples.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings in decedent offspring included, no milk in stomach, abdominal contents autolysed, animal thin and cannabalized head and forelimbs.
On Day 13 of Age (scheduled termination) one litter at 10000 ppm had pups that were yellow in color; no effect of treatment is inferred.

Histopathological findings:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 332 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

The mean concentrations were within 3% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Conclusions:

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity, reproductive performance and survival of the offspring is 10000 ppm (Toxicity males: 551 mg/kg/day, Toxicity females: 625 mg/kg/day, Females during gestation: 609 mg/kg/day and females during lactation: 1332 mg/kg/day).
The reductions in offspring body weight gain were similar or less than those seen in the adults and therefore the NOAEL for offspring growth and development is also 10000 ppm (1332 mg/kg/day during lactation).

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in compliance with GLP. Maderal ZA1076 was administered to Sprague Dawley rats by dietary administration for at least six weeks. An additional subgroup was also used to assess reversibility, persistence or delayed occurrence of systemic effects for at least 28 days post treatment. Females within the toxicity and recovery groups were not paired. Males within the recovery groups were also not paired.

The study design was as follows:

Group	Treatment	Dietary concentration (ppm)	Reproductive phase	Number of toxicity phase animals		Number of recovery animals
			Females	Male	Female	Male	Female
1	Control	0	10	10	5	5	5
2	MADERAL ZA1076	1000	10	10	5	-	-
3	MADERAL ZA1076	3000	10	10	5	-	-
4	MADERAL ZA1076	10000	10	10	5	5	5

 

Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks.

Recovery phase animals were treated for six weeks followed by a 28-day recovery period.

Reproductive phase females were treated for two weeks before pairing, throughout pairing and gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. Selected F1 offspring were sampled and killed on Day 4 (not analyzed) or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age.

Similarly constituted Control groups were assigned to each phase and received untreated basal diet for the same duration.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, 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, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

The achieved dosages for the Toxicity and Recovery phase animals during treatment and for reproductive phase females before pairing (F0) were 54.9, 169 and 551 mg/kg/day for males and 65.4, 214 and 625 mg/kg/day for females, at dietary concentrations of 1000, 3000 or 10000 ppm respectively. 

The achieved dosages for reproductive phase females during gestation were 61.5, 197 and 609 mg/kg day, and during lactation were 142, 427 and 1332 mg/kg/day, at dietary concentrations of 1000, 3000 or 10000 ppm respectively.

The mean concentrations were within 3% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis.  The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

The general appearance, behaviour, sensory reactivity, grip strength and motor activity of the animals were unaffected by treatment.

The T4 concentrations in the offspring were comparable with endogenous levels in all treatment groups, and the T4 concentrations in the adult males were not significantly reduced; therefore, no effect of treatment is inferred.

Group mean food intake was reduced in all groups receiving treated diet during the first week of treatment and was reduced in toxicity phase females receiving 1000 or 10000 ppm throughout the remainder of the treatment period. During gestation and lactation, food intake was reduced at 10000 ppm. The reductions in food consumption were considered to be most likely caused by an effect of the test item on the palatability of the diet.

 
After a four-week recovery period, food consumption for males and females previously treated with 10000 ppm was similar to the Controls.

Reduced body weight gain was seen during the first week of treatment for males receiving 3000 or 10000 ppm, and a body weight loss was seen during this period in females receiving 10000 ppm.  Body weight gain remained reduced for the reproductive phase females treated with 10000 ppm during the first half of the gestation phase and during the lactation phase. The reductions in bodyweight gain were considered to be most likely caused by the reduced food consumption.

Body weight gain for males and females previously treated with 10000 ppm was increased during recovery when compared with the Controls; hence displaying initial signs of recovery from any treatment related body weight effect.  By the end of the four week recovery period, the mean body weight of the males previously treated with 10000 ppm was similar to the mean body weight of the Control males, and the mean body weight of the females previously treated with 10000 ppm was slightly reduced when compared with the mean body weight of the Control females.

All reproductive phase females were pregnant and successfully littered down; litter size, post implantation survival index, live birth index, viability index and sex ratio were all considered to be unaffected by treatment.

There was no effect of treatment on ano-genital distance in male or female offspring, and males did not have nipples at any dose.

The body weight gain of offspring from mothers treated with 10000 ppm was reduced compared to controls (19% lower than in Controls); due to the lower food consumption in the mothers.

In Week 7 for both sexes treated with 10000 ppm, the white blood cell, neutrophil, lymphocyte, eosinophil, monocyte count and APTT were all slightly low when compared with the control group.  Large unstained cells were high in all treated groups of males.  The haematocrit and mean cell hemoglobin in females during Week 7 were low; this effect was not seen in the males.  On Day 13 of lactation similar parameters were affected by treatment in females receiving 1000 or 10000 ppm; white blood cell, neutrophil, lymphocyte and eosinophil count and APTT were all low. 

By the end of recovery for males and females previously treated with 10000 ppm, white blood cell and lymphocyte counts were all increased when compared with the Controls.  Large unstained cells remained slightly high in males after four weeks of recovery, neutrophil count was increased and APTT remained low in females previously treated with 10000 ppm when compared with Controls. 

The biochemical examination in Week 7, revealed high plasma urea concentrations, high plasma cholesterol and high triglyceride concentration in both sexes receiving 10000 ppm.  Increased plasma glucose concentrations were seen in all treated groups of males.  Low phosphorus concentrations were seen in both sexes receiving 3000 or 10000 ppm; attaining statistical significance in females receiving 10000 ppm.  Slightly increased total protein concentration, increased albumin and globulin were seen in toxicity phase animals receiving 3000 or 10000 ppm.  Chloride concentrations in males treated with 10000 ppm was also low.  Plasma urea, cholesterol and triglyceride concentrations were comparable with the Control group after four weeks of recovery in males and females previously treated with 10000 ppm.  Low phosphorus concentrations remained in both sexes after four weeks of recovery, but chloride concentrations in males following four weeks of recovery was similar to the plasma chloride concentrations in Control males, and protein, albumin and globulin concentrations were generally similar to Controls. On Day 13 of lactation, plasma urea concentrations, plasma glucose concentrations, plasma cholesterol and triglyceride concentrations displayed the same treatment related effects as seen in the toxicity animals.  However, no major differences between treated animals and the Controls were seen in phosphorus, total protein concentrations and albumin or globulin concentrations. 

Following six weeks of treatment and when compared with the Control, absolute and adjusted liver weights were increased in both sexes receiving 3000 or 10000 ppm.  The absolute and adjusted heart weights were marginally reduced in both sexes receiving 10000 ppm.  After four weeks of recovery, the absolute and adjusted liver weights for both sexes were only marginally increased in the animals previously treated with 10000 ppm, when compared with the Controls.  Hence, displaying some degree of recovery from the treatment related effect on the liver; such effects are usually considered to be non-adverse adaptive responses.

There were no macropathology changes considered to be related to treatment.

Histopathological changes related to treatment with MADERAL ZA1076 at 10000 ppm were seen in the liver, thyroids and adrenals of males and females and the kidneys of males: in the liver, hypertrophy of centrilobular hepatocytes was seen in all males and females treated with 10000 ppm and accounted for the higher than control group mean body weight adjusted liver weight for males and females of this dose group.  Following the 4-week recovery period, centrilobular hepatocellular hypertrophy was not seen in any male or female that was previously treated with 10000 ppm and, in consequence, demonstrated full recovery in both sexes.

In the thyroids, follicular cell hypertrophy/hyperplasia was seen at higher incidences in males and females treated with 10000 ppm than in the respective control animals; complete recovery from this change was seen in both sexes. These effects are considered to be not adverse.

In the adrenals, diffuse hypertrophy of the zona glomerulosa was seen in the majority of males and females treated with 10000 ppm; complete recovery form this change was seen in both sexes. These effects are considered to be not adverse.

In the kidneys of males, an accumulation of hyaline droplets in the cortical tubular epithelium was seen in all animals treated with 10000 ppm.  An accumulation of hyaline droplets was seen in one male of each of the groups treated with 1000 or 3000 ppm respectively: this change showed full reversal after 4 weeks recovery.   Basophilia of cortical tubules was seen in the majority of males treated with 10000 ppm and in one control male only a marginal degree of recovery in terms of incidence was evident for this change following 4 weeks recovery. Such effects are specific to the male rat kidney and are considered to be not relevant to humans.

  

Conclusion

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity, reproductive performance and survival of the offspring is 10 000 ppm (Toxicity males: 551 mg/kg/day, Toxicity females: 625 mg/kg/day, Females during gestation: 609 mg/kg/day and females during lactation: 1332 mg/kg/day).

The reductions in offspring body weight gain were similar or less than those seen in the adults and therefore the NOAEL for offspring growth and development is also 10 000 ppm (1332 mg/kg/day during lactation).

 

The reductions in body weight gain are considered to be related to the reduced food intake consumed during lactation by the adult females, which was likely caused by the reduced palatability of the diet resulting from the inclusion of the test item. However, as this is a screening study it cannot be excluded that there is an alternative mechanism for the observed effect on weight gain, but there are no correlates in the biochemistry or histopathology data, so this can only be speculation.

 

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirement for an OECD 408 study in rats. 

 

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2021-02-12

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

EC Number:

815-500-1

Cas Number:

1853175-99-1

Molecular formula:

C15 H26 O2

IUPAC Name:

2-(3,3-dimethylcyclohex-1-en-1-yl)-2,5,5-trimethyl-1,3-dioxane

Test material form:

liquid

Details on test material:

- Physical state: Colorless liquid
- Storage condition of test material: Store in cool (ambient temperature) and dry place

Test animals

Species:

rat

Strain:

Crj: 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) rat 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 nulliparous and non-pregnant: yes
- Age at study initiation: Males 71 to 77 days old / Females 85 to 92 days old
- Weight at study initiation: Males 326 to 406 g / Females 224 to 296 g
- Fasting period before study: no
- 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
Pre-pairing, toxicity phase females and recovery animals: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: one female
Lactation: one female + litter
- Diet (e.g. ad libitum): Non-restricted SDS VRF1 Certified powdered diet
- Water (e.g. ad libitum): Non-restricted. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 8 days (Males) / 22 days (Females)
- Environmental enrichment: A soft white untreated wood block and a plastic shelter were provided to each cage throughout the study (except during lactation) and replaced when necessary or at the same time as the cages. In addition, approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation and changed at the same frequency as cage bedding.


DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-09-04 To: 2019-12-11

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The most appropriate route of administration was evaluated according to REACH Regulation. The oral route of administration via dietary inclusion was selected as it will maximize the systemic availability (internal dose).

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): SDS VRF1 Certified powdered diet
- Storage temperature of food: At ambient temperature (15 to 25°C) for up to 24 days.
- Method of preparation: The test item was incorporated into the plain 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 item was weighed into a suitable container. An amount of diet that approximately equaled the weight of test item was added and the mixture stirred together. A further amount of diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder, after which it was made up to the final weight of the premix with diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.

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.
In this study the homogeneity and stability of MADERAL ZA1076 in the diet matrix was demonstrated for up to 24 days when stored at ambient (15 to 25°C) or frozen temperature (-10 to -30°C).
- Achieved concentration:
Samples of each formulation prepared for administration in Week 1 of treatment and on Day 12 of lactation were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks.
Recovery phase animals were treated for six weeks followed by a 28-day recovery period.
Reproductive phase females were treated for two weeks before pairing, throughout pairing and gestation and until Day 12 of lactation.

Frequency of treatment:

Continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 ppm

Remarks:

54.9 mg/kg/day for males and 65.4 mg/kg/day for females

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

169 mg/kg/day for males and 214 mg/kg/day for females

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Remarks:

551 mg/kg/day for males and 625 mg/kg/day for females

No. of animals per sex per dose:

- Reproductive phase: 10 Females
- Toxicity phase: 10 Males / 5 Females
- Recovery: 5 Males and 5 Females (control and high dose groups)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations selected for this OECD 422 study based on the results of a 14-day preliminary toxicity study.
In the preliminary toxicity study, dietary concentrations of 1500, 5000 and 15000 ppm were investigated. Males receiving 15000 showed low body weight gain between Days 1 and 4, but good weight gain thereafter. After 14 Days of feeding of treated diet the group mean body weight was similar for males treated with 1500, 5000 or 15000 ppm Maderal ZA1076. For females fed with diet containing 15000 ppm group mean body weight loss was apparent up until Day 4 of treatment, a general improvement in body weight performance was then seen in females for the remainder of the treatment period. Overall females had a small group mean body weight loss by Day 15 of treatment.
A reduction in food intake was observed from Day 1-4 of treatment in both males and females fed treated diet containing 15000 ppm. An improvement in food consumption was observed throughout the remainder of the treatment period, such that food consumption was similar throughout the groups by the end of the first week of treatment. There were no test item related macroscopic findings. Both males receiving 15000 ppm and females receiving 5000 or 15000 ppm had increased liver weights. All other organ weights were similar throughout the groups.
Based on the results of this preliminary toxicity study, a high dietary level of 10000 ppm an intermediate level of 3000ppm, and a low level of 1000 ppm were selected for the Main study.
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: used to assess reversibility, persistence or delayed occurrence of systemic effects
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, on Days 0, 7, 14 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.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization (not reported).
Before feeding of the treated diets on the day that treatment commenced and weekly thereafter (including recovery phase) and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization (not reported).
Before feeding of the treated diets on the day that treatment commenced and weekly before pairing.
Days 0, 7, 14, and 20 after mating and Days 1, 4, 7, and 13 of lactation.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Days 22 to 28). Food consumption was recorded continuously for Toxicity phase females and Recovery phase animals.
For Reproductive phase females after mating food consumption was recorded to match the body weight schedule: Days 0-6, 7-13, 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination, or on Day 13 of lactation (lactating females)
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: No
- How many animals: The five lowest numbered surviving Toxicity phase males per group. All Toxicity phase females. The first five lactating females with a litter per group (Reproductive phase). All Recovery phase males and females.
- Parameters checked in table 7.6.1/1 were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: at termination, or on Day 13 of lactation (lactating females)
- Animals fasted: No
- How many animals: The five lowest numbered surviving Toxicity phase males per group. All Toxicity phase females. The first five lactating females with a litter per group (Reproductive phase). All Recovery phase males and females.
- Parameters checked in table 7.6.1/1 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Parameters examined: Thyroid stimulating hormone (TSH) and Thyroxine (T4) from sublingual vein blood sample site from an isoflurane anesthesia.
- Time of blood sample collection and how many animals:
At termination: All surviving F0 males (Toxicity and Recovery phase) and Toxicity and Recovery phase females. All surviving F0 Reproductive phase females.
Day 4 of age: up to two females per litter (where possible)
Day 13 of age: two males and two females per litter (where possible)
- Animals fasted: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: on the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 5 of treatment and on the first five lactating main study females in each group at Days 7-9 of lactation.
- Battery of functions tested: sensory activity and grip strength (approach response, pinna reflex, auditory startle reflex and tail pinch response) / motor activity

IMMUNOLOGY: No

OTHER: Cf. IUCLID 7.8.1
- Estrous cycle
- Mating procedure
- Parturition observation and gestation length

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.6.1/2-3-4)

HISTOPATHOLOGY: Yes (see table 7.6.1/2-3-4)

Optional endpoint(s):

Optional endpoints: No

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non significant, are presented on the relevant tables. For some parameters, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption during the treatment phase and the before pairing phase for Reproductive phase females were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during treatment, gestation and lactation
Hematology
Blood chemistry
Estrous cycles during treatment
Pre-coital interval
Gestation length
Stage of estrous cycle at termination (Toxicity phase)
Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index,
live birth index and viability index), for before blood sampling study periods
Ano-genital distance, adjusted for Day 1 pup body weight
Organ weights, absolute and adjusted for terminal body weight

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

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

The sequence of statistical tests used for each parameters were detailed below.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no findings during the detailed physical examination and arena investigations associated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in males fed 3000 or 10000 ppm MADERAL ZA1076 (80% of the Controls for both groups) during the first week of treatment: and slightly reduced in all treated groups from Day 22-29. Toxicity phase females fed diet containing 10000 ppm showed minor weight loss in first week of treatment, weight gain remained reduced during Days 15-43 of treatment. Reproductive females treated with 10000 ppm prior to pairing had reduced body weight gain prior to pairing and during gestation (Days 0-7, 7-14, and 14-20), when compared with the Controls. At the beginning of the recovery period, males and females previously treated with 10000 ppm had a reduced group mean body weight when compared with the Control group. Body weight gain for males and females previously treated with 10000 ppm was increased when compared with the Controls; hence displaying initial signs of recovery from any treatment related body weight effect. By the end of the four-week recovery period, the mean body weight of the males previously treated with 10000 ppm was similar to the mean body weight of the Control males, and the mean body weight of the females previously treated with 10000 ppm was slightly reduced when compared with the mean body weight of the Control females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food intake was reduced in all groups receiving treated diet during the first week of treatment and remained reduced in toxicity phase females receiving 1000 or 10000 ppm throughout the remainder of the treatment period. During treatment and prior to pairing females fed 10000 ppm had a reduced group mean food intake when compared with the controls. These females continued to have reduced food consumption throughout gestation and lactation, respectively. Food consumption for males and females previously treated with 10000 ppm was similar to the controls for the last three weeks of recovery, food consumption for previously treated females during the first week of recovery was reduced when compared with the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematological examination in Week 7 did identify differences from the control that were likely to be attributable to treatment; therefore further investigation during recovery was instigated.
In Week 7 for both sexes treated with 10000 ppm, the white blood cell, neutrophil, lymphocyte, eosinophil, monocyte count and APTT were all reduced when compared with the control group. Large unstained cells were increased in all treated groups of males. The haematocrit and mean cell hemoglobin in females during Week 7 were reduced; this effect was not seen in the males. Further investigation to determine any recovery from these effects was performed on these parameters.
By the end of recovery for males and females previously treated with 10000 ppm, white blood cell and lymphocyte counts were all increased when compared with the Controls. Large unstained cells remained slightly increased in males after four weeks of recovery, neutrophil count was increased and APTT remained reduced in females previously treated with 10000 ppm when compared with Controls.
On Day 13 of lactation similar parameters were affected by treatment in females receiving 1000 or 10000 ppm; white blood cell, neutrophil, lymphocyte and eosinophil count and APTT were all reduced. However, females treated with 3000 ppm generally had marginally increased or similar values to the controls for these parameters.
All other differences from control were minor or did not show relationship to dose or confined to one sex, and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination in Week 7, revealed increased plasma urea concentrations, increased plasma cholesterol and triglyceride concentrations in both sexes receiving 10000 ppm. Increased plasma glucose concentrations were seen in all treated groups of males. Reduced phosphorus concentrations were seen in both sexes receiving 3000 or 10000 ppm. Slightly increased total protein concentration, increased albumin and globulin were seen in toxicity phase animals receiving 3000 or 10000 ppm. Chloride concentrations in males treated with 10000 ppm was also reduced. Further investigation to determine any recovery from these effects was performed on these parameters. Plasma urea, cholesterol and triglyceride concentrations were comparable with the Control group after four weeks of recovery in males and females previously treated with 10000 ppm. Reduced phosphorus concentrations remained in both sexes after four weeks of recovery, but chloride concentrations in males following four weeks of recovery were similar to the plasma chloride concentrations in Control males, and protein, albumin and globulin concentrations were generally similar to Controls.
On Day 13 of lactation, plasma urea concentrations, plasma glucose concentrations, plasma cholesterol and triglyceride concentrations displayed the same treatment related effects as seen in the toxicity animals. However, no major differences between treated animals and the controls were seen in phosphorus, total protein concentrations and albumin or globulin concentrations. Alkaline phosphatase and alanine transferase activities were low in all treated groups; attaining statistical significance in all treated groups for alanine transferase and females treated with 10000 ppm for alkaline phosphatase. However, a reduction in enzyme activity is generally considered non adverse and of no toxicological importance. Bile acid concentrations were increased for females receiving 10000 ppm; however, there was considerable inter-group variation and the difference did not achieve statistical significance. Sodium and chloride concentrations were slightly high at 3000 or 10000 ppm.

Endocrine findings:

no effects observed

Description (incidence and severity):

The T4 concentrations in the offspring were comparable with endogenous levels in all treatment groups, and the T4 concentrations in the adult males were not significantly reduced; therefore, no effect of treatment is inferred.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Sensory Reactivity Observations and Grip Strength:
There were no changes in sensory reactivity or grip strength attributable to treatment.
When compared with the control, hindlimb grip strength was low in females on Day 7-9 of lactation receiving 1000 ppm. However, as there is no dose relationship this is likely attributed to natural variation and is therefore not an effect of treatment; hence, no sensory reactivity or grip strength investigations were performed during recovery.
- Motor activity:
Group mean activity scores for all groups of treated males during Week 5 are low when compared with the controls, with several 18 and 36 minute high and low beam interval scores achieving statistical significance in the males. All but one of these scores are within the HCD range; so no effect of treatment is inferred. There was no effect of treatment on the activity of toxicity phase females at 10000 ppm. Females receiving 3000 or 10000 ppm on Day 7-9 of lactation have decreased high and low mean beam scores; all of which are within the HCD range. Due to these scores being within the HCD range, there was not considered to be a treatment related effect and therefore motor activity investigations were not performed during recovery.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following six weeks of treatment and when compared with the control, absolute and adjusted liver weights were increased in both sexes receiving 3000 or 10000 ppm. The absolute and adjusted heart weights were marginally reduced in both sexes receiving 10000 ppm.
On Day 13 of lactation, the absolute and adjusted weights of the uterus, cervix and oviducts were marginally reduced for all treated groups; with a dose response apparent for adjusted weights.
After four weeks of recovery, the absolute and adjusted liver weights for both sexes were only marginally increased in the animals previously treated with 10000 ppm, when compared with the controls. Hence, displaying effects of recovery from the treatment related effect on the liver. However, for both sexes previously treated with 10000 ppm, the thyroid and parathyroid weight was slightly reduced at the scheduled termination during recovery; whereas the thyroids and parathyroids had a slightly increased weight following six weeks of treatment in those treated with 10000 ppm.
All other differences in organ weights were minor, only evident in a single sex, or failed to show a relationship with dietary concentration and were therefore considered to be unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings at macroscopic examination of the toxicity phase animals, the reproductive phase females or the recovery phase animals that were considered attributable to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1/ Animals Killed After 6 Weeks of Treatment
Changes related to treatment with MADERAL ZA1076 at 10000 ppm were seen in the liver, thyroids and adrenals of males and females and the kidneys of males.
- Liver: Minimal centrilobular hepatocellular hypertrophy was seen in all animals treated with 10000 ppm.
- Thyroids: Minimal follicular cell hypertrophy/hyperplasia was seen at a higher incidence in males and females treated with 10000 ppm than in the respective control animals.
- Adrenals: Minimal diffuse hypertrophy of the zona glomerulosa was seen in the majority of animals treated with 10000 ppm.
- Kidneys: A minimal to moderate accumulation of hyaline droplets in the cortical tubular epithelium was seen in all males treated with 10000 ppm. The kidneys of animals treated with 1000 or 3000 ppm were only examined if macroscopic changes were seen at necropsy. At microscopy, minimal and slight accumulation of hyaline droplets was seen in one male of the groups treated with 1000 or 3000 ppm respectively. Minimal or slight basophilia of cortical tubules was seen in the majority of males treated with 10000 ppm and at the minimal degree in one control male.

2/ Animals Killed After 4 Weeks of Recovery
Kidneys: A minimal accumulation of hyaline droplets in the cortical tubular epithelium was seen in one male control. Minimal or slight basophilia of cortical tubules was seen in the majority of males that were previously treated with 10000 ppm and at the minimal degree in one control male.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Cf. IUCLID section 7.8.1 for results linked to reproductive and developmental toxicity

Details on results:

The mean concentrations were within 3% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity females

Effect level:

>= 625 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 551 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effect observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity, reproductive performance and survival of the offspring is 10000 ppm (Toxicity males: 551 mg/kg/day, Toxicity females: 625 mg/kg/day)

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in compliance with GLP. Maderal ZA1076 was administered to Sprague Dawley rats by dietary administration for at least six weeks. An additional subgroup was also used to assess reversibility, persistence or delayed occurrence of systemic effects for at least 28 days post treatment. Females within the toxicity and recovery groups were not paired. Males within the recovery groups were also not paired.

The study design was as follows:

Group	Treatment	Dietary concentration (ppm)	Reproductive phase	Number of toxicity phase animals		Number of recovery animals
			Females	Male	Female	Male	Female
1	Control	0	10	10	5	5	5
2	MADERAL ZA1076	1000	10	10	5	-	-
3	MADERAL ZA1076	3000	10	10	5	-	-
4	MADERAL ZA1076	10000	10	10	5	5	5

 

Toxicity phase males were treated for two weeks before pairing up to necropsy after six weeks. Toxicity phase females were treated for six weeks.

Recovery phase animals were treated for six weeks followed by a 28-day recovery period.

Reproductive phase females were treated for two weeks before pairing, throughout pairing and gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy). The offspring received no direct administration of the test item; any exposure was in utero or via the milk. Selected F1 offspring were sampled and killed on Day 4 (not analyzed) or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age.

Similarly constituted Control groups were assigned to each phase and received untreated basal diet for the same duration.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, 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, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

The achieved dosages for the Toxicity and Recovery phase animals during treatment and for reproductive phase females before pairing (F0) were 54.9, 169 and 551 mg/kg/day for males and 65.4, 214 and 625 mg/kg/day for females, at dietary concentrations of 1000, 3000 or 10000 ppm respectively. 

The achieved dosages for reproductive phase females during gestation were 61.5, 197 and 609 mg/kg day, and during lactation were 142, 427 and 1332 mg/kg/day, at dietary concentrations of 1000, 3000 or 10000 ppm respectively.

The mean concentrations were within 3% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis.  The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

The general appearance, behaviour, sensory reactivity, grip strength and motor activity of the animals were unaffected by treatment.

The T4 concentrations in the offspring were comparable with endogenous levels in all treatment groups, and the T4 concentrations in the adult males were not significantly reduced; therefore, no effect of treatment is inferred.

Group mean food intake was reduced in all groups receiving treated diet during the first week of treatment and was reduced in toxicity phase females receiving 1000 or 10000 ppm throughout the remainder of the treatment period. During gestation and lactation, food intake was reduced at 10000 ppm. The reductions in food consumption were considered to be most likely caused by an effect of the test item on the palatability of the diet.

 
After a four-week recovery period, food consumption for males and females previously treated with 10000 ppm was similar to the Controls.

Reduced body weight gain was seen during the first week of treatment for males receiving 3000 or 10000 ppm, and a body weight loss was seen during this period in females receiving 10000 ppm.  Body weight gain remained reduced for the reproductive phase females treated with 10000 ppm during the first half of the gestation phase and during the lactation phase. The reductions in bodyweight gain were considered to be most likely caused by the reduced food consumption.

Body weight gain for males and females previously treated with 10000 ppm was increased during recovery when compared with the Controls; hence displaying initial signs of recovery from any treatment related body weight effect.  By the end of the four week recovery period, the mean body weight of the males previously treated with 10000 ppm was similar to the mean body weight of the Control males, and the mean body weight of the females previously treated with 10000 ppm was slightly reduced when compared with the mean body weight of the Control females.

All reproductive phase females were pregnant and successfully littered down; litter size, post implantation survival index, live birth index, viability index and sex ratio were all considered to be unaffected by treatment.

There was no effect of treatment on ano-genital distance in male or female offspring, and males did not have nipples at any dose.

The body weight gain of offspring from mothers treated with 10000 ppm was reduced compared to controls (19% lower than in Controls); due to the lower food consumption in the mothers.

In Week 7 for both sexes treated with 10000 ppm, the white blood cell, neutrophil, lymphocyte, eosinophil, monocyte count and APTT were all slightly low when compared with the control group.  Large unstained cells were high in all treated groups of males.  The haematocrit and mean cell hemoglobin in females during Week 7 were low; this effect was not seen in the males.  On Day 13 of lactation similar parameters were affected by treatment in females receiving 1000 or 10000 ppm; white blood cell, neutrophil, lymphocyte and eosinophil count and APTT were all low. 

By the end of recovery for males and females previously treated with 10000 ppm, white blood cell and lymphocyte counts were all increased when compared with the Controls.  Large unstained cells remained slightly high in males after four weeks of recovery, neutrophil count was increased and APTT remained low in females previously treated with 10000 ppm when compared with Controls. 

The biochemical examination in Week 7, revealed high plasma urea concentrations, high plasma cholesterol and high triglyceride concentration in both sexes receiving 10000 ppm.  Increased plasma glucose concentrations were seen in all treated groups of males.  Low phosphorus concentrations were seen in both sexes receiving 3000 or 10000 ppm; attaining statistical significance in females receiving 10000 ppm.  Slightly increased total protein concentration, increased albumin and globulin were seen in toxicity phase animals receiving 3000 or 10000 ppm.  Chloride concentrations in males treated with 10000 ppm was also low.  Plasma urea, cholesterol and triglyceride concentrations were comparable with the Control group after four weeks of recovery in males and females previously treated with 10000 ppm.  Low phosphorus concentrations remained in both sexes after four weeks of recovery, but chloride concentrations in males following four weeks of recovery was similar to the plasma chloride concentrations in Control males, and protein, albumin and globulin concentrations were generally similar to Controls. On Day 13 of lactation, plasma urea concentrations, plasma glucose concentrations, plasma cholesterol and triglyceride concentrations displayed the same treatment related effects as seen in the toxicity animals.  However, no major differences between treated animals and the Controls were seen in phosphorus, total protein concentrations and albumin or globulin concentrations. 

Following six weeks of treatment and when compared with the Control, absolute and adjusted liver weights were increased in both sexes receiving 3000 or 10000 ppm.  The absolute and adjusted heart weights were marginally reduced in both sexes receiving 10000 ppm.  After four weeks of recovery, the absolute and adjusted liver weights for both sexes were only marginally increased in the animals previously treated with 10000 ppm, when compared with the Controls.  Hence, displaying some degree of recovery from the treatment related effect on the liver; such effects are usually considered to be non-adverse adaptive responses.

There were no macropathology changes considered to be related to treatment.

Histopathological changes related to treatment with MADERAL ZA1076 at 10000 ppm were seen in the liver, thyroids and adrenals of males and females and the kidneys of males: in the liver, hypertrophy of centrilobular hepatocytes was seen in all males and females treated with 10000 ppm and accounted for the higher than control group mean body weight adjusted liver weight for males and females of this dose group.  Following the 4-week recovery period, centrilobular hepatocellular hypertrophy was not seen in any male or female that was previously treated with 10000 ppm and, in consequence, demonstrated full recovery in both sexes.

In the thyroids, follicular cell hypertrophy/hyperplasia was seen at higher incidences in males and females treated with 10000 ppm than in the respective control animals; complete recovery from this change was seen in both sexes. These effects are considered to be not adverse.

In the adrenals, diffuse hypertrophy of the zona glomerulosa was seen in the majority of males and females treated with 10000 ppm; complete recovery form this change was seen in both sexes. These effects are considered to be not adverse.

In the kidneys of males, an accumulation of hyaline droplets in the cortical tubular epithelium was seen in all animals treated with 10000 ppm.  An accumulation of hyaline droplets was seen in one male of each of the groups treated with 1000 or 3000 ppm respectively: this change showed full reversal after 4 weeks recovery.   Basophilia of cortical tubules was seen in the majority of males treated with 10000 ppm and in one control male only a marginal degree of recovery in terms of incidence was evident for this change following 4 weeks recovery. Such effects are specific to the male rat kidney and are considered to be not relevant to humans.  

 

Conclusion

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity, reproductive performance and survival of the offspring is 10 000 ppm (Toxicity males: 551 mg/kg/day, Toxicity females: 625 mg/kg/day, Females during gestation: 609 mg/kg/day and females during lactation: 1332 mg/kg/day).

The reductions in offspring body weight gain were similar or less than those seen in the adults and therefore the NOAEL for offspring growth and development is also 10 000 ppm (1332 mg/kg/day during lactation).

 

The reductions in body weight gain are considered to be related to the reduced food intake consumed during lactation by the adult females, which was likely caused by the reduced palatability of the diet resulting from the inclusion of the test item. However, as this is a screening study it cannot be excluded that there is an alternative mechanism for the observed effect on weight gain, but there are no correlates in the biochemistry or histopathology data, so this can only be speculation.

 

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirement for an OECD 422 study in rats.