2-tert-butyl-1,4-dimethoxybenzene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 422 (Rat)

NOAEL:

Systemic Toxicity (males): 150 mg/Kg bw/day (based on effects on body weight and body weight gain)

Systemic Toxicity (females): 450 mg/Kg bw/day

Reproductive Toxicity: 450 mg/Kg bw/day

Developmental Toxicity: 450 mg/Kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-07 to 2019-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations did not adversely affect the results or integrity of the complete study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Emerald Kalama Chemical Limited (Dans Road, WA8 0RF Widnes, Cheshire, United Kingdom); Batch/Lot number: A180118C
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-07-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤ 70% relative humidity), under inert gas, protected from humidity (stored in a tightly closed container).
- Stability under storage conditions: stable for 15 days when stored at room temperature
- Stability under test conditions: test material was stable in the selected vehicle in the 15-220 mg/mL concentration range for 15 days at room temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: stable in the selected vehicle in the 15-220 mg/mL concentration range for 15 days

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, almost colourless liquid

OTHER SPECIFICS
- other information: Purity: 99.78%

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar)

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in
toxicity and reproduction toxicity studies and known fertility. The same strain was used for the Dose Range Finding study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, Sulzfeld, D-97633, Germany), from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approximately 11-12 weeks old at the start of the experiment, 14-15 weeks old at the start of the treatment and 16-17 weeks old at mating
- Weight at study initiation: (P) Males: 478-562 g; Females: 234–316 g
- Fasting period before study: Not specified
- Housing: Group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively.
- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 639 38520, Expiry date: April 2019)
produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-24.2°C (target range 22 ± 3 °C)
- Humidity (%): 20 – 49 % (target range 30-70 %)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2018-11-15 To: 2019-02-21

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400 (PEG 400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle (as a clear solution) in the Pharmacy of the Test Facility. Formulations were prepared daily (fresh prior to administration to animals) at appropriate concentrations (according to the dose level and treatment volume selected) in the Pharmacy of the Test Facility as follows:

A calculated amount of the test material was added into a beaker, which was then filled up to the
calculated final volume with the vehicle. The mixture was mixed vigorously by a magnetic stirrer to make a homogenous formulation (solution) and kept mixed until the end of treatment, as applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of a short solubility test performed at the Test Facility earlier, polyethylene glycol 400 (PEG 400) was selected as vehicle for this study in agreement with the Sponsor based on the formulation and analytical trials. The same vehicle was used in the Acute Oral Toxicity study and in the Dose Range Finding (DRF) study.
- Concentration in vehicle: 0, 10, 30, or 90 mg/mL for the control, low dose, mid dose, and high dose groups, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg bw
- Lot/batch no. (if required): ACROS; Batch number: A0401858 / A0392628
- Purity: Not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occurred (the mating period lasted for 5 days)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified but females remained with the same male until copulation occurred
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method (High Performance Liquid Chromatography with ultraviolet detection) at the Test Site for analytical work. Top, middle and bottom duplicate samples (0.5 mL per replicate) were taken from test material formulations three times during the study (during the first week of treatment, midway during the treatment period, and once in the last 8 days); one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. As an additional fourth sampling set, the remaining dose formulations of the last treatment day were used.

Formulation samples were put into 1.5 polyethylene or 2-mL polypropylene tubes and their weight was recorded for the first three sampling occasions as per Study Plan. Due to one out of range Low dose result, an additional measurement was made from samples taken on the last treatment day (when all dose groups were treated).

Formulation samples were kept at room temperature until shipment. Samples (both sets) were shipped at room temperature as soon as possible after collection for concentration and/or homogeneity measurement to the Principal Investigator of the Test Site for analytical work. Formulation analysis was conducted within the determined stability period under the control of the Principal Investigator in compliance with the Study Plan, its amendment and/or the relevant SOPs of the Test Site for analytical work.

Acceptance criteria of the concentration analysis were set according to the analytical method validation; it was 100 ± 10 % of the mean nominal concentration. Acceptance criteria of the homogeneity was that the coefficient of variation (CV) of replicates (top, middle and bottom of test item formulations) should be less than 10%.

Duration of treatment / exposure:

Dosing of both sexes was performed for 2 weeks before mating, during mating, and continued up to and including the day before the necropsy.

Males: dosed for 28 days (14 days pre-mating and 14 days mating/post-mating)

Females: dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing).

Frequency of treatment:

Once daily, 7 days/week, for 2 weeks before mating, during mating, and continued up to and including the day before the necropsy.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control (Polyethylene glycol 400 (PEG 400))

Dose / conc.:

50 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

150 mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

450 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels for this OECD 422 study were selected by the Sponsor in consultation with the Study Director based on available data from the acute oral toxicity study (Test Facility Study code: 17/183-001P, LD50 > 2000 mg/Kg bw in rats) and results of the Dose Range Finding (DRF) study performed at the Test Facility (Test Facility Study code: 17/181-220PE). In the preliminary study, treatment for one week at 1000 mg/Kg/day resulted in a liver weight increase of about 60%, which was considered not compatible with a longer term study such as OECD No. 422. Consequently, a maximum dose level of 450 mg/Kg bw/day was selected in consultation with the Sponsor, with the aim of inducing toxic effects, but ideally no death or suffering. The oral route was selected as it is one of the possible routes of human exposure.

- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges on the day of start of treatment. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the computer software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

- Fasting period before blood sampling for clinical biochemistry: Yes. Animals were fasted (overnight period of food deprivation, in case of dams it meant the night after the litter had been culled).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General observations were performed once a day (No general clinical observations were made at the start, on those days when detailed clinical observations were made and on the day of necropsy), during the pre-treatment and treatment period (in the afternoon (pm) or after treatment at approximately the same time with minor variations as practical during the working day), as no peak period of effects was noted after dosing during the first few days of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then weekly (in the morning (am) or before treatment) and before necropsy. These observations were performed outside the home cage in a standard arena, at similar day times as practical. Animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed at least weekly during the preexposure period, on Day 0 for randomization purposes (prior to first exposure), afterwards at least weekly and at termination.

Parental females were weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the parental female animals measured on GD 3, 10 and 17 plus PPD 10 were measured additionally, only for the calculation of accurate individual gavage treatment volumes but not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Animal food consumption was determined by re-weighing the non-consumed diet on the days of body weight measurements. The daily intake per animal was calculated (During pairing, while one male and one female were in a single cage, the food intake was measured, but data was not statistically analyzed since it is not appropriate to use the mean value, because males eat more than females) for statistical data analysis and reporting.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OTHER:
- Functional observation battery (FOB) and locomotor activity measurement:
Five males and five females of the parental generation were randomly selected in each group for these measurements.

Assessment of potential test material related neurotoxicity was performed during the last exposure week (males on Day 24; females on PPD8-10). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.

To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. This step was performed 3 times for each animal.

Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test material. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal. The procedure was repeated with the hind limbs (3 times for each animal) and the appropriate grip support.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.

Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmia, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalization were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.

Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field of 50 x 50 cm for a 1-hour observation time; a DVD recording of activity/movement was made. A recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions for each evaluated animal. The DVD was analyzed with “SMART” software after all recordings were made, to produce the appropriate parameters. The data from all groups was evaluated for total travelled distance in 5-minute segments of the 1 hour. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

- Observation of the delivery process:
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded. The duration of
gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition. Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

- Clinical Pathology:
All adult animals, including the selected animals for blood sampling (5 animals per sex per group), were fasted (overnight period of food deprivation, in case of dams it meant the night after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times: The parameters listed in Table 2 were evauated for selected animals.
Clinical chemistry: The parameters listed in Table 3 were evauated for selected animals.

- Urinalysis:
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours. The evaluation of the urine samples was performed as indicated in Table 4. Due to the observed brown discolouration of the High and Mid dose male urine samples as determined by visual assessment at necropsy, absorbance (λ= 405 nm) of a small aliquot (200 μL) was measured for each male by a plate reader. Remaining urine samples were additionally frozen at -20°C for possible future analysis.

- Thyroid Hormone Analysis:
For thyroid hormone analysis, blood samples were taken by venepuncture from vena sublingualis (in case of parental animals) or by decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows: from up to two pups per litter on PND4; from all dams at termination (PPD14) and up to two pups per litter on PND13; from all adult males at termination. Pup blood (plasma) was pooled by litter.

Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4ºC). The resulting plasma was divided in at least two aliquots (volume target of at least 125 μL for the first aliquot and at least 75 μL for the second aliquot, if possible; any leftover material was also retained for safety reason as a third aliquot) and stored in an ultrafreezer (-80 ±10°C) until shipping for analysis.

Samples of adult males and PND13 pups were assessed for T4 levels. The measurement of the T4 hormone levels in adult females and PND4 pups, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary as per Study Plan. Thyroid hormone analysis was conducted using a validated method under the control of the Principal Investigator in compliance with Study Plan, its amendment and the relevant SOPs of the Test Site.

T4 measurement was based on a solid phase extraction from plasma followed by an analysis of the extract by reverse phase liquid chromatography (HPLC) with MS/MS detection after ionization using an Electro Spray Interface (ESI, positive mode). The analytical procedure for the determination of T4 in rat plasma by LC-MS/MS was developed at the Charles River Laboratories Evreux (formerly Citoxlab France). Complete validation was carried out prior to the analysis of the study samples. The validated concentration range was 4.00 to 500 ng/mL for T4.

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for each female (all females including spare animals) by vaginal smears daily for 15 days during the pre-exposure period. Females that failed to show a 4 or 5-day cycle was not included in the study. Twelve females showing regular oestrus cycles were treated and used for mating in each group (the rest of the females were moved back to the spare colony after the mating process had been finished). Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized or autolyzed) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured by a calliper at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13 (individual records were maintained).

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4. All surviving pups were terminated on PND13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified

Postmortem examinations (parental animals):

SACRIFICE
At termination, the surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination. The anaesthetic product was diluted for pups’ euthanasia as required.

GROSS NECROPSY
Gross necropsy was performed on all adult animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.

Vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs. The number of implantation sites and of corpora lutea were recorded in females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 5 and Table 6 weighed and prepared for microscopic examination , respectively.

In case of samples for microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For adult animals, a detailed histological examination was performed as follows:

• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group)
• found dead High dose female (#4502)
• organs / tissues of all animals where macroscopic findings (abnormalities) were seen at necropsy,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males (#2003, #3005 and #3007) that failed to sire and all females (#2503, #3505 and #3507) that failed to deliver healthy pups
• on the liver and thymus of remaining females from the Control, Low, Mid and High dose groups (due to test item-related finding the selected High dose females).

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow). Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

Dead pups and pups euthanized on PND4 or PND13 were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive organs which were examined for signs of altered development. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs. Presence of nipples/areolae in PND13 pups was also recorded.

Statistics:

For information on statistical analysis please see 'Any other information on materials and methods incl. tables'.

Reproductive indices:

1) Male Mating Index (%): (Number of males with confirmed mating / Total Number of males cohabited) x 100

2) Female Mating Index (%): (Number of sperm − positive females / Total Number of females cohabited) x 100

3) Male Fertility Index (%): (Number of males impregnating a female / Total Number of males cohabited) x 100

4) Female Fertility Index (%): (Number of pregnant females / Number of sperm - positive females) x 100

5) Gestation Index (%): (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

1) Survival Index (%): (Number of live pups (at designated time) / Number of pups born) x 100

2) Pre-implantation mortality (%): (Number of corpora lutea-Number of implantations / Number of corpora lutea) x 100

3) Intrauterine mortality (%): (Number of implantations-Number of liveborns / Number of implantations) x 100

4) Total mortality (%): (Number of implantations-Number of viable pups (PND0/4/13) / Number of implantations) x 100

5) Sex ratio % (male/females): (Number of male / female pups (PND0/4/13) / Number of pups examined (PND0/4/13)) x 100

Pre-implantation mortality and intrauterine mortality were calculated together as pre-natal mortality in relevant tables. The Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed through the study period.

One Control male (#1004) displayed piloerection from Day 18 to Day 28. It should be noted that during treatment on Day 13, this animal bit off part of the plastic gavage tube and a small part of the tube remained in the GI tract. That piece of plastic (80 mm long) was present in the stomach / duodenum at the time of scheduled necropsy (Day 28). This fact is considered to have contributed to the observed findings in the later period.

Hunched back (on Day 42) and piloerection (between Days 40 and 53) were observed in one low dose female (#2508). However, these findings might be related to the parturition / lactation period (the animal delivered on GD 22 (i.e. Day 39), but all her pups died within one day).

A nodule (1-2 cm) in the abdominal area was observed for one mid dose female (#3511) on Days 39-41. Noisy respiration was observed for a high dose female (#4502) on Day 7. This animal was found dead later, confirmed as a gavage error.

All the above observations were considered to either be incidental or technical issue-related, and not related to the treatment with the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality was observed through the study period. One High dose female (#4502) was found dead on Day 13. However, as a 2x1 mm perforation was identified on the thoracic part of the oesophagus at necropsy, this fact was clearly caused by a technical error (gavage accident), not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related reduced body weight gain was noted in high dose males, but no such effect was observed in male rats in the mid- and low dose groups. A slight body weight loss was recorded for high dose males in the first week of treatment when controls gained about 9 grams (the difference to control was
statistically significant at p<0.05). Although animals showed some recovery later in the treatment period, the body weight gain calculated for the entire treatment period of the high dose males was reduced when compared to control males (by approximately 28%, the difference was not statistically significant).

Exposure to the test material did not impact body weight or body weight gain in female rats when compared with control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment-related adverse effects in the mean daily food consumption were observed in any treated group when compared to the controls. Although slightly decreased food consumption (statistically significant at p<0.05) was observed in the first week of the treatment in high dose males (by 5%) when compared to control, the difference was minor and the observed value was in line with the general historical control data of this strain. Additionally, there was no statistically significant difference in the daily food consumption value of high dose males compared to controls when calculated for the entire treatment period. No similar trend was observed in female rats. While the effect does correspond with the body weights of the high dose male rats in the first week, this minor transient difference was not considered to be an an adverse treatment-related effect. Any other occasional statistically significant differences were regarded as incidental and of no toxicological significance.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences or biologically relevant treatment-related changes were observed in males and females of the treated groups when compared with control animals.

Dose related increase in the white blood cell count was observed in treated male and female animals when compared to the control group (20% in high dose males, and 44% in high dose females). However, no statistical significance was gained and all the individual values were within the historical control range. Therefore, no clear treatment-related effect could be concluded.

The relative amount of eosinophil leukocytes showed relatively large percentage differences in the treated groups when compared to controls due to the low absolute values which is normal for this parameter. The same trend was observed in the treated males and females, but there was no statistical significance in any of those cases. The observed values were within the historical control range, thus, these differences were considered as animal variability, and not attributed to treatment with the test material. No statistically significant and biologically relevant changes were recorded for any other haematology parameters in the treated male and female rats.

There were no significant changes or biologically relevant effects on the measured coagulation parameters that could be ascribed to treatment with the test material. A statistically significant decrease in the prothrombin time was detected in the female animals of all dose groups (at p<0.05) compared to control. However, all the mean values were within the historical control range, there was no dose response, and no similar trend was observed in the male animals. Therefore, it was not considered to be a treatment-related effect.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant changes or biologically relevant effects on serum chemistry that could be ascribed to the test material.

A statistically significant increase (at p<0.05) in the albumin concentration (by approx. 9%) was detected in high dose males; and statistically significant increases (at p<0.05) in the potassium and calcium concentration (by 13% and 6%, respectively) were detected in high dose females when compared to the relevant control animals. However, the observed values were well within the historical control range, there were no clear dose response in any of those cases, and no similar trends were observed in the other sex. Thus, these differences were not considered to be treatment-related.

Slight statistically significant differences (p<0.05) in the urea concentration of the low dose females were considered incidental due to the lack of dose response and biological relevance. No other statistically significant changes were recorded for any other parameters in the treated male and female rats.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes were observed in any of the treated groups when compared to the control.

Significantly (at p<0.01) lower pH values of the collected urine samples were recorded in the high and mid dose males when compared to the control. However, the observed mean values were comparable with the pH of control females, and all the individual values were within the historical control range. No similar trend was observed in female animals (although a slight decrease in high and low dose females was observed). Therefore, the observed differences were not considered to be a treatment-related effect.

Slight increase (significant at p<0.05) in the semi-quantitative glucose assessment were also observed in the high and mid dose males when compared to the control animals. However, the observed values were comparable with the female data and no similar trend was observed in female animals. Therefore, the observed differences were not considered to be a treatment-related effect.

A clear difference in the urinary colour was observed: light brown or dark brown colour was observed in mid dose and high dose males, respectively. The visual assessment was confirmed by absorbance measurement of the samples. The observed discolouration was treatment-related, but not considered as an adverse effect. No similar findings were noted at the necropsy of the female animals.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related neurological effects were observed as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or treated groups. No statistically significant or biologically relevant differences were observed during the assessment of landing foot splay test or grip strength in any treated group (males and females) when compared to the control group.

Male and female rats of all dose groups had a normal locomotor activity. In all cases, the initial activity was high, with generally a reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. There was no statistically significant difference between the treated animals and the relevant control groups when evaluating the total travelled distance for the entire period of 0-60 minutes.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Parental Generation (Found Dead)
The histopathological examination of the high dose female ((#4502) found dead on Day 13 confirmed correlating findings including focal perforation of the oesophagus, mild focal haemorrhage and minimal multifocal congestion in the lungs, mild multifocal congestion and minimal neutrophilic infiltrate in the ovaries. In addition, mild focal epicardial inflammation of the heart, minimal decreased of lymphocytes of the thymic cortex and minimal eosinophilic infiltrate in the glandular stomach were seen.

Parental Generation (Non-pregnant)
Organs from the reproductive system (ovary, oviduct, uterus, cervix and vagina) of three non-pregnant females: one female in the low dose group (#2503), and two females in the mid dose group (#3505 and #3507) were microscopically examined. The examination did not reveal any treatment-related findings. Reproductive organs (testes, epididymis, prostate, seminal vesicle and coagulating gland) of their mating partners (#2003, #3005 and #3007 males) were also examined microscopically. No treatment-related changes were detected in any of those cases, although minimal, multifocal degeneration / atrophy of testis was noted for #2003.

Parental Generation (Terminal - Males: Days 28; Females: PPD14)
Treatment-related minimal centrilobular hepatocellular hypertrophy was observed in 9/11 examined females of the high dose group (#4501, #4504, #4505, #4506, #4507, #4508, #4510, #4511 and #4512). This microscopic finding correlated with increased organ weight changes. Track down evaluation of mid and low dose group samples showed no similar findings in the liver.

Some other changes were seen in control and treated animals without meaningful differences in severity and incidence, and were considered to be incidental or a common background. These included minimal bilateral vacuolation of the adrenal gland cortex recorded in two control males (#1002 and #1009) and two high dose males (#4001 and #4005). Diffuse / multifocal hypertrophy (minimal) in the zona glomerulosa of the adrenals was observed in two control females (#1503 and #1507) and in one high dose female (#4501). Minimal/mild infiltration of inflammatory cells in the caecum were observed in two control females (#1507 and #1508) and in one high dose female (#4506).

Moderate necrotizing inflammation was observed in the oesophagus of one control male
(#1004). A mild multifocal haemorrhage in the adventitia and mild erythrophagocytosis and haemorrhage as well as infiltration of inflammatory cells in the lung-associated lymph nodes were also recorded in this animal. These observations correlated with the necropsy.

Minimal bilateral retinal atrophy was observed in the eyes of one high dose female (#4506). Presence of porphyrin (bilateral / unilateral) in the eye (Harderian gland) was observed in one control male (#1010), one control female (#1508), and in two high dose male rats (#4007 and #4009). Unilateral / bilateral metaplasia (minimal) was observed in the lachrymal glands of one control male (#1010), three high dose males (#4001, #4005 and #4007) and two high dose females (#4501 and #4512).

Minimal tubular basophilia (unilateral, focal/multifocal) was seen in the kidney of one control male (#1010) and in two high dose males (#4007 and #4009). Minimal hyaline cast (unilateral/bilateral, focal/multifocal) was observed in the kidney of one control male (#1002), three high dose males (#4005, #4007 and #4009) and in one control female (#1507). Mild pyelonephritis (bilateral) with presence of bacteria was observed in one low dose female (#2508), this fact correlated with the necropsy. Interstitial fibrosis (bilateral, focal) was also recorded for this animal. Mineralization (minimal) was present in the pelvis or corticomedullary junction of the kidney in one control female (#1508) and in one high dose female (#4506).

Minimal multifocal infiltration of inflammatory cells was seen in the liver of one control male (#1009) and one low dose female (#2508). Severe torsion in the liver of a control female (#1509) correlated with necropsy. Hepatocellular vacuolation was recorded in the liver of a control female (#1508). Mild pulmonary haemorrhage was detected in one control male (#1001) and in one high dose male (#4003). These observations correlated with the necropsy.

Minimal unilateral / bilateral erythrophagocytosis was recorded in the mandibular lymph nodes of one control male (#1005) and in one control female (#1509). Minimal infiltration of inflammatory cells was detected in the pancreas of three high dose males (#4001, #4005 and #4007). A single cyst was present in the pituitary glands of a high dose female (#4505). This finding correlated with the observation recorded at necropsy.

Atrophy was observed in the prostate of one control male (#1002) rat and in one high dose male
(#4004). Minimal / mild infiltration of inflammatory cells in the prostate was also recorded in five control male rats (#1005, #1006, #1009, #1011 and #1012), in one low dose male (#2003), and in eight high dose males (#4001, #4002, #4004, #4005, #4007, #4010, #4011 and #4012). Minimal focal haemorrhage in the prostate was present in one mid dose male (#3005) and in one high dose male (#4012) rat.

Mild dilatation of the spinal cord was detected in one control male (#1009) while minimal extramedullary haematopoiesis was observed in the spleen of one control male (#1002), two Control females (#1503 and #1509), and two high dose male rats (#4001 and #4005).

Infiltration of inflammatory cell in the glandular submucosa of the stomach was observed in two control males (#1008 and #1010), one control female (#1509), three high dose males (#4003, #4007 and #4009), and in one high dose female rats (#4501).

Decreased size or cellularity was observed in the thymus of one control male (#1002), two high dose males (#4003 and #4007), and in one high dose female (#4505). Hypertrophy (minimal) in the follicular cells of thyroids was detected in one control female (#1508) and in one high dose female (#4506).

Neutrophilic infiltrate (minimal / mild) in the tongue was observed in one control male rat (#1005), two control female rats (#1506 and #1509), two high dose male rats (#4001 and #4003), and in one high dose female (#4504). Mild / minimal infiltration of inflammatory cells was seen in the vagina of four control females (#1504, #1505, #1508 and #1510) and six high dose females (#4504, #4507, #4508, #4509, #4510 and #4512). Degeneration / atrophy of testis (multifocal, minimal) was noted in one low dose male (#2003), exfoliation of germ cells and presence of multinucleated giant cells was also recorded in this male. Minimal cell debris was also present in the epididymis of this male animal. Additionally, the presence of multinucleated giant cells was observed in a high dose male (#4001). Minimal epithelial vacuolation (focal) of the epididymis was seen in another high dose male rat (#4010). Infiltration of inflammatory cells in the inguinal mammary gland area of one high dose female (#4505) was also recorded.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effect of the test material was observed in the study based on the results of thyroid hormone analysis. Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related effect on oestrus cycles was observed in the study. Each female selected for the treatment showed acceptable cycles (mean cycle length was 4.00 days for each group) before starting the treatment period. There was no treatment-related effect on the oestrus cycle of females (mean cycle length was in the 4.00-4.03 days for each group after the treatment). No prolonged oestrus was recorded in any of the treated groups. Prolonged dioestrus was observed for one high dose female (#4502), but this frequency (1/12) was in line with the normal, expected range. One female in the low dose group (#2509) and one female in the mid dose group (#3506) showed pseudopregnancy, but both of them was successfully mated later.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and treated groups with regard to reproductive ability, mating, fertility or gestation.

The mating indices (males and females) were 100% in all groups. There was one non-pregnant female in the low dose group (#2503) and two non-pregnant females (#3505 and #3507) in the mid dose group. The fertility indices (males and females) were 100%, 92%, 83%, and 100% for the control, low, mid, and high dose groups, respectively while the gestation index was 100% in all groups.

Exposure to the test material was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation) for all females in the treated group. The mean duration of mating was 2.67, 2.42, 2.83, and 2.73 days in control, low, mid, and high dose groups, respectively.

No effect of treatment was noted during the gestation, parturition and post-partum period. The mean length of gestation was 22.17, 22.18, 22.60 and 22.18 days in the control, low, mid, and high dose groups, respectively. Although there was a statistical difference (p<0.05) in the gestation length of the mid dose group when compared to control data, all the individual data were in the range of 22-23 days, therefore in line with the normal physiological range. There was no dose response, thus these results were considered as not indicating any treatment-related effect.

During the study, parturition was normal for almost all animals while abnormal delivery (longer than 5 hours) was noted for one high dose female (#4510). The number of implantation sites was comparable to the control mean in all dose groups and no statistically significant differences were observed.

There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal, or total mortality values (litter mean and %) in any dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Systemic Toxicity

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Based on external evaluation, no clinical signs or abnormalities were recorded for any of the pups except for one mid dose male pup (#3511/9), where alterations on the genitals, (misshapen genital tubercle, i.e. additional tags of tissue on the genital tubercle), cysts on the testis, and large right testis were observed. These findings were considered as minor based on the confirmation of the Foetal Specialist of the Test Facility.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no treatment-related mortality or effect on the survival of pups observed in the study. Evidence of suckling was recorded for all live born pups in the study.

There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups. Slightly higher number of pups died in the lactation period in the Low dose group, but 25 out of the 28 animals in this group belonged to two dams (#2508 and #2509). No living pups were in those litters by PND1 or PND3, respectively. There was no dose response, and this observation was considered as animal variability, unrelated to treatment.

The number of viable pups on PND0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group. Thus there was no treatment-related effect on the viability of pups at those time points.

The ratio of female pups was slightly higher in the mid and low dose groups than in the control group, but as the obtained data were within the historical control range, there was no statistical significance and no dose response, this finding was attributed to biological variability and not related to the treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related differences in the offspring body weights or weight gains were observed in any treated group when compared to the controls.

When evaluated per litter basis, the mean litter body weights on PND 0, PND 4, and PND 13, and body weight gain in the relevant periods showed no statistically significant differences compared to controls in the F1 generation (the difference was less than 5% in every case).

Food consumption and compound intake (if feeding study):

not specified

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 specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No treatment-related effect was observed on anogenital distance during the study.

No statistically significant changes in the anogenital distance measured on PND 0 were observed for treated male and female pups when litter mean values were compared to control values.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed on nipple retention during the study. No nipples/areolae were counted for any male pups on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 Generation (Found Dead)
No treatment-related macroscopic findings were observed in unscheduled deaths of F1 pups. There were no stillborn pups in the study. Cannibalization of 5 control, 19 low dose, 0 mid dose, and 5 high dose pups was observed at birth or in the lactation period. One autolyzed pup was also observed in the mid dose group. There were no stillborn or intact found dead pups in any groups. In the lactation period, a total of 16 autolyzed pups were found in the Control group and 13 in the low dose groups. No special findings were recorded for those pups. The cause of death was not established for any pups.

F1 Generation (Terminal - PND13)
No treatment-related macroscopic findings were recorded in F1 offspring generation euthanized and examined externally at scheduled termination on PND13. In particular, no alteration in the development of external reproductive organs was observed. The presence of internal sex organs was also checked.

Histopathological findings:

not examined

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive / Developmental Toxicity

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 7. Analytical Results
Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of the nominal Concentration (%)
Analytical sampling #1 (Sampling: 03 January 2019)
Control	<LOQ	N/A
10	9.93 ± 0.12	99.3
30	31.26 ± 0.71	104.2
90	93.19 ± 1.20	103.5
Analytical sampling #2 (Sampling: 23 January 2019)
Control	<LOQ	N/A
10	9.78 ± 0.18	97.8
30	28.76 ± 1.77	95.9
90	90.14 ± 1.59	100.2
Analytical sampling #3 (Sampling: 13 February 2019) Set #1
Control	<LOQ	N/A
10	20.08^§± 1.44	200.8
30	27.09 ± 1.03	90.3
90	84.54 ± 2.99	93.9
Analytical sampling #3 (Sampling: 13 February 2019) Set #2
Control	<LOQ	N/A
10	18.02^§± 5.88	180.2
30	30.64 ± 0.86	102.1
90	95.50 ± 6.30	106.1
Analytical sampling #4 (Sampling: 18 February 2019)
Control	<LOQ	N/A
10	9.39 ± 0.16	93.9
30	29.85 ± 0.98	99.5
90	83.93 ± 0.97	93.3

Notes: Mean measured concentrations with the 95% confidence intervals are shown.

LOQ (Limit of Quantification) was 3 μg/mL for analytical samples, which equalled to 0.6 mg/mL for formulation samples.

N/A: Not applicable

§: value was out of the acceptance criteria

Table 8. Summary of Selected Body Weight Parameters of Parental Animals
Parameter	Dose Groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
Male, Body weight on Day 27 (g) (NS)	549.6	544.8	546.3	539.8
	Difference (%)	-0.9	-0.6	-1.8
Male, Body weight gain Day 0-7 (g) (DN)	8.7	6.5	6.9	-0.7*
	Difference (%)	-25.0	-20.2	-107.7
Male, Body weight gain Day 0-27 (g) (NS)	26.7	25.3	25.8	19.3
	Difference (%)	-5.2	-3.7	-27.7
Females
Female, Body weight on GD20 (g) (NS)	437.3	445.0	444.0	447.6
	Difference (%)	1.8	1.5	2.4
Female, Body weight on PPD13 (g) (NS)	381.4	385.8	398.6	392.5
	Difference (%)	1.2	4.5	2.9
Female, Body weight gain Day 0-PPD13 (g) (NS)	98.2	102.0	119.0	109.0
	Difference (%)	3.9	21.2	11.0

Notes:

Data (group mean values, n=10-12) were rounded to one decimal place. Non-pregnant (#2503, #3505 and #3507) or found dead (#4502) females as well as one control male (#1004) were excluded from the evaluation

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Test

NS: Statistically not significant when compared to the control

 

Table 9. Results of Daily Food Consumption (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Male, Food consumption Day 0-7 (g) (DU)	28.02	28.13	27.36	26.57*
	difference (%)	0.4	-2.4	-5.2
Male, Food consumption Day 0-27 (g) (NS)	27.62	28.00	27.61	27.25
	difference (%)	1.4	0.0	-1.3
Female, Food consumption Day 0-7 (g) (DU)	17.99	20.13**	21.10**	19.43
	difference (%)	11.9	17.3	8.0
Female, Food consumption GD 0-20 (g) (NS)	26.05	27.53	27.42	27.25
	difference (%)	5.7	5.2	4.6
Female, Food consumption PPD 0-13 (g) (NS)	54.71	52.22	59.91	57.69
	difference (%)	-4.5	9.5	5.4

Notes:

Data (group mean values, n=10-12) were rounded to two decimal places

Non-pregnant (#2503, #3505 and #3507) or found dead (#4502) females were excluded from the

evaluation.

GD: Gestation Day, PPD: Post-partum Day.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Test

DU: Dunn test

NS: Statistically not significant when compared to the control.

Data from cage level (group mean values, n=5) were rounded to two decimal places.

 

Table 10. Results of selected FOB and SMART parameters (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
Landing foot splay, mm (hind paws) (NS) HC range: 26-129	86.9	103.8	99.9	98.1
	difference (%)	19.5	15.0	13.0
Grip-strength, g (forelimbs) (NS) HC range: 1100-2333	1861.1	2031.1	1831.1	1885.5
	difference (%)	9.1	-1.6	1.3
Grip-strength, g (hind limbs) (NS) HC range: 483-1378	913.1	926.9	878.7	840.5
	difference (%)	1.5	-3.8	-8.0
Total travelled distance (cm) (NS) HC data: 2307-10078	6511.3	7036.9	6687.8	7378.1
	difference (%)	8.1	2.7	13.3
Females
Landing foot splay, mm (NS) (hind paws) HC range: 35-108	85.8	73.3	74.1	71.5
	difference (%)	-14.5	13.7	-16.6
Grip-strength, g (forelimbs) (NS) HC range: 795-1935	1487.4	1631.1	1438.1	1581.1
	difference (%)	9.7	-3.3	6.3
Grip-strength, g (hind limbs) (NS) HC range: 392-1265	373.3	412.5	388.8	428.1
	difference (%)	10.5	4.1	14.7
Total travelled distance (cm) (NS) HC data: 2749-11632	7062.5	9443.1	7260.9	6498.5
	difference (%)	33.7	2.8	-8.0

Notes:

Data (group mean values, n=5) are rounded to one digit, HC data are rounded to whole number

Total travelled distance of 0-60 minutes was calculated

HC: Historical control

NS: Statistically not significant when compared to the control.

 

Table 11. Results of Selected Haematology and Coagulation Parameters (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
White Blood Cell count (10^9/L) (NS) HC range: 0.90-10.35	2.88	3.09	3.28	3.45
	difference (%)	7.6	14.0	20.0
Relative amount of Eosinophils (%) (NS) HC range: 0.00-3.90	2.56	2.02	1.76	1.80
	difference (%)	-21.1	-31.3	-29.7
Prothrombin time (seconds) (NS) HC range: 9.2-10.6	9.72	9.44	9.46	9.66
	difference (%)	-2.9	-2.7	-0.6
Females
White Blood Cell count (10^9/L) (NS) HC range: 0.61-8.03	2.08	2.55	2.62	2.99
	difference (%)	22.7	26.1	43.7
Relative amount of Eosinophils (%) (NS) HC range: 0.30-9.30	1.80	1.90	1.66	1.54
	difference (%)	5.6	-7.8	-14.4
Prothrombin time (seconds) (DN) HC range: 7.4-9.9	9.04	8.82*	8.80*	8.80*
	difference (%)	-2.4	-2.7	-2.7

Notes: Data (group mean values, n=5) were rounded to two decimal places

HC: Historical control

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Test

NS: Statistically not significant compared to control

 

Table 12. Results of Selected Clinical Chemistry Parameters (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
Albumin (g/L) (DN) HC range: 23.10-35.60	31.44	32.80	32.68	34.10*
	difference (%)	4.3	3.9	8.5
Potassium (mmol/L) (NS) HC range: 4.30-7.70	5.92	5.74	6.08	6.00
	difference (%)	-3.0	2.7	1.4
Calcium (mmol/L) (NS) HC range: 2.28-2.76	2.64	2.69	2.67	2.70
	difference (%)	1.9	1.1	2.4
Females
Albumin (g/L) (NS) HC range: 25.70-42.70	26.36	28.26	28.48	28.48
	difference (%)	7.2	8.0	8.0
Potassium (mmol/L) (DN) HC range: 4.00-7.40	6.16	6.80	6.04	6.96*
	difference (%)	10.4	-1.9	13.0
Calcium (mmol/L) (DN) HC range: 2.36-2.96	2.54	2.55	2.48	2.70*
	difference (%)	0.5	-2.5	6.1

Notes: Data (group mean values, n=5) were rounded to two decimal places

HC: Historical control

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Test

NS: Statistically not significant when compared to the control

 

Table 13. Results of Selected Urinalysis Parameters (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
pH (DU) HC range: 6.0-8.0	8.0	7.8	6.4**	6.4**
	difference (%)	-2.5	-20.0	-20.0
Glucose (semi-quantitative) (DU)	0.0	0.6	1.2*	1.4*
	difference (%)	N/AP	N/AP	N/AP
Females
pH (NS) HC range: 6.0-8.0	6.4	6.0	6.2	6.0
	difference (%)	-6.3	-3.1	-6.3
Glucose (semi-quantitative) (NS)	0.8	1.8	0.8	0.8
	difference (%)	N/AP	N/AP	N/AP

Notes: Data (group mean values, n=5) were rounded to one decimal place

HC: Historical control

N/AP: Not applicable for semi-quantitative assessment

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DU: Dunn test

NS: Statistically not significant when compared to control

 

Table 14. Results of Reproductive Parameters (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	0
Number of males used for mating	12	12	12	11^§
Number of successful mating	12	12	12	11
Number of fertile males	12	11	10	11
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	92	83	100
Females
Number of treated animals	12	12	12	12
Number of pre-terminal death	0	0	0	1^§
Number of females used for mating	12	12	12	11
Number of sperm-positive females	12	12	12	11
Number of females with no implantation sites	0	1	2	0
Number of pregnant females	12	11	10	11
Pregnant females with live born(s)	12	11	10	11
Pregnant females not delivered living pups	0	0	0	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	92	83	100
Female gestation index (%)	100	100	100	100

§Note: One female in the High dose group (#4502) was found dead before the mating started. Therefore, its male pair was not used for mating.

 

Table 15. Results of Pregnancy Evaluation (P Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Number of evaluated females	12	12	12	11
Number of pregnant females	12	11	10	11
Duration of pregnancy (days) (D)	22.17	22.18	22.60*	22.18
Number of implantations, mean (NS)	15.33	13.92	13.50	15.82
Number of pups born, mean (NS)	14.33	14.27	14.40	14.55
Number of live born pups, mean (NS)	14.08	13.91	14.30	14.45
Pre-natal mortality, mean (NS)	1.25	1.27	1.90	1.36
Pre-natal mortality (%), mean (NS)	7.75	7.77	11.10	8.62
Post-natal mortality, mean (NS)	1.50	2.55	0.00	0.36
Post-natal mortality (%), mean (NS)	9.53	20.00	0.00	2.36
Total mortality, mean (NS)	2.75	3.82	1.90	1.73
Total mortality (%), mean (NS)	16.35	23.42	11.10	10.85

Notes:

Data (group mean values) were rounded to two decimal places. Pre-natal mortality includes

intrauterine mortality and the loss at delivery. Post-natal mortality PND0-13 and total mortality on

PND13 are shown in the table (the number of pups culled for blood sampling on PND4 were

excluded).

Statistical significance compared to control: * = p<0.05, ** = p<0.01

D: Duncan’s Multiple Range Test

NS: Statistically not significant when compared to the control

 

Table 16. Summary of Survival (Offspring – F1)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Number of evaluated litters	12	11	10	11
Number of pups born, mean (NS)	14.33	14.27	14.40	14.55
Number of live born pups, mean (NS)	14.08	13.91	14.30	14.45
Number of living pups on PND13, mean (NS)	10.75	9.73	12.50	12.09
Pre-natal mortality, mean (NS)	1.25	1.27	1.90	1.36
Pre-natal mortality (%), mean (NS)	7.75	7.77	11.10	8.62
Post-natal mortality on PND0-4, mean (NS)	1.42	2.45	0.00	0.36
Post-natal mortality on PND0-4 (%), mean (NS)	8.89	19.39	0.00	2.36
Total mortality on PND4, mean (NS)	2.67	3.73	1.90	1.73
Total mortality on PND4 (%), mean (NS)	15.71	22.82	11.10	10.85
Pups culled for blood sampling, mean (NS)	1.83	1.64	1.80	2.00
Post-natal mortality on PND0-13, mean (NS)	1.50	2.55	0.00	0.36
Post-natal mortality on PND0-13 (%), mean (NS)	9.53	20.00	0.00	2.36
Total mortality on PND13, mean (NS)	2.75	3.82	1.90	1.73
Total mortality on PND13 (%), mean (NS)	16.35	23.42	11.10	10.85
Survival index on PND0 (NS)	98.19	97.35	99.33	99.24
Sex ratio (%) on PND0 (NS)	43.65	55.94	51.98	45.49
Survival index on PND4 (NS)	91.11	80.61	100.00	97.64
Sex ratio (%) on PND4 (NS)	43.38	53.98	51.98	45.54
Survival index on PND13 (NS)	99.17	99.15	100.00	100.00
Sex ratio (%) on PND13 (NS)	44.01	52.19	50.19	44.49

Notes:

Data (group mean values) were rounded to two decimal places. Sex ratio means the percentage of females per litter. Culling for blood sampling was made on PND4. Survival index was calculated in comparison with the end of previous period (on PND0 it was compared to the number of pups born, on PND4 it was compared to the number of live born pups, on PND13 it was compared to the number of pups after culling on PND4).

NS: Statistically not significant when compared to control

 

Table 17. Summary of Mortality (Offspring – F1)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Number of evaluated litters	12	11	10	11
Number of pups born(NS)	172 / 12	157 / 11	144 / 10	160 / 11
Number of cannibalized pups (NS)	3 / 3	4 / 3	0 / 0	1 / 1
Number of autolyzed pups (NS)	0 / 0	0 / 0	1 / 1	0 / 0
Number of stillborn pups (NS)	0 / 0	0 / 0	0 / 0	0 / 0
Number of live born pups(NS)	169 / 12	153 / 11	143 / 10	159 / 11
Number of found dead pups (born alive) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Number of living pups on PND0(NA)	169 / 12	153 / 11	143 / 10	159 / 12
Number of cannibalized pups (PND0-13) (CH)	2 / 2	15** / 5	0 / 0	4 / 4
Number of autolyzed pups (PND0-13) (NA)	16 / 2	13 / 1	0 / 0	0 / 0
Number of found dead, intact pups (PND0-13) (NA)	0 / 0	0 / 0	0 / 0	0 / 0
Total number of pups died (born alive) (NA)	18 / 3	28 / 5	0 / 0	4 / 4
Culled for blood sampling on PND4 (NA)	22 / 11	18 / 9	18 / 9	22 / 11
Number of viable pups on PND13(NS)	129 / 11	107 / 9	125 / 11	133 / 11

Notes:

Mortality numbers mean number of pups / number of affected litters. PND0-13 means the

lactation period, counted after the delivery was ended

Statistical significance compared to control: * = p<0.05, ** = p<0.01 (negative trends were not taken

into evaluation)

NA: Not applicable

CH: Chi square test

NS: Statistically not significant compared to control

 

Table 18. Summary of Selected Body Weight Parameters of Offspring (F1)
Parameter	Dose Groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Number of evaluated litters	12	11	10	11
Mean litter body weight (PND0), g (NS)	6.29	6.23	6.43	6.33
Mean litter body weight (PND4), g (NS)	10.50	10.78	10.84	10.70
Mean litter body weight gain (PND0-4), g (NS)	4.20	4.33	4.41	4.37
Mean litter body weight (PND13), g (NS)	29.90	30.00	29.52	29.25
Mean litter body weight gain (PND4-13), g (NS)	19.42	19.18	18.67	18.54
Mean litter body weight gain (PND0-13), g (NS)	23.59	23.53	23.08	22.92

Notes: Body weight / body weight gain data (litter mean values) were rounded to two decimal places

NS: Statistically not significant when compared to control

 

Table 19. Anogenital Distance (Offspring (F1))
Parameter	Dose Groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Male Pups
Number of evaluated litters	12	11	10	11
Number of evaluated male pups (NS)	95	68	70	87
Anogenital distance, litter mean of males (mm) (NS)	3.10	2.99	3.18	3.02
Anogenital distance, all male pups (mm) (NA)	3.08	2.99	3.18	3.04
Minimum / Maximum value, litter mean (mm)	2.8 / 3.3	2.7 / 3.4	2.9 / 3.6	2.6 / 3.3
Minimum / Maximum value, all males (mm)	2.47 / 3.77	2.21 / 3.89	2.50 / 3.95	2.33 / 3.97
Female Pups
Number of evaluated litters	12	11	10	11
Number of evaluated female pups (NS)	74	85	73	72
Anogenital distance, litter mean of females (mm) (NS)	1.70	1.68	1.67	1.65
Anogenital distance, all female pups (mm) (NA)	1.71	1.69	1.68	1.64
Minimum / Maximum value, litter mean (mm)	1.5 / 2.0	1.6 / 1.9	1.4 / 1.8	1.3 / 1.9
Minimum / Maximum value, all females (mm)	1.17 / 2.33	1.13 / 2.33	1.19 / 2.19	1.18 / 2.27

Notes:

Data (group mean or litter mean values) were rounded to one or two decimal places

NS: Statistically not significant when compared to control

NA: Not applicable

 

Table 20. Results of Selected Organ Weights (Parental Generation)
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Males
Terminal body weight, g (NS)	525.4	518.6	524.5	520.4
	difference (%)	-1.3	-0.2	-0.9
Liver (absolute), g (DN)	14.201	15.278	16.515**	18.497**
	difference (%)	7.6	16.3	30.3
Liver (relative to body), % (DN)	2.748	2.944	3.148**	3.559**
	difference (%)	7.2	14.6	29.5
Thymus (absolute), g (NS)	0.366	0.299	0.357	0.339
	difference (%)	-18.2	-2.5	-7.3
Thymus (relative to body), % (NS)	0.071	0.058	0.068	0.065
	difference (%)	-18.4	-4.0	-8.1
Thyroid (absolute), g (NS)	0.0286	0.0280	0.0270	0.0280
	difference (%)	-2.0	-5.5	-2.0
Thyroid (relative to body), % (NS)	0.0054	0.0054	0.0052	0.0054
	difference (%)	-0.2	-4.6	-0.3
Females
Terminal body weight, g (NS)	342.0	351.6	357.8	359.4
	difference (%)	2.8	4.6	5.1
Liver (absolute), g (DN)	13.339	13.771	14.891*	15.802**
	difference (%)	3.2	11.6	18.5
Liver (relative to body), % (DN)	3.900	3.903	4.163	4.402**
	difference (%)	0.1	6.8	12.9
Thymus (absolute), g (DU)	0.271	0.233	0.229	0.188**
	difference (%)	-14.1	-15.4	-30.4
Thymus (relative to body), % (DU)	0.080	0.067	0.064	0.052**
	difference (%)	-16.5	-19.6	-34.3
Thyroid (absolute), g (NS)	0.0233	0.0210	0.0225	0.0221
	difference (%)	-10.0	-3.6	-5.3
Thyroid (relative to body), % (NS)	0.0068	0.0060	0.0063	0.0062
	difference (%)	-12.4	-7.9	-9.3

Notes:

Data (group mean values, n=10-12) were rounded to one, three or four decimal places

Thyroid and parathyroid weights were measured together

Statistical significance compared to control: * = p<0.05, ** = p<0.01

D: Dunnett’s Multiple Range Test

DU: Dunn test

NS: Statistically not significant when compared to the control

 

Table 21. Selected Parameters related to Thyroid Hormone Levels
Parameter	Dose groups
	Control (0 mg/Kg/day)	50 mg/Kg/day	150 mg/Kg/day	450 mg/Kg/day
Parental Males
Number of evaluated males	12	12	12	12
T4 concentration (ng/mL) (NS) HC range: 34.3-60.7	44.08	43.39	46.56	42.83
	difference (%)	-1.6	5.6	-2.8
Thyroid gland weights (g) (NS) HC range: 0.009-0.059	0.0286	0.0280	0.0270	0.0280
	difference (%)	-2.0	-5.5	-2.0
Thyroid gland / body weight (%) (NS) HC range: 0.0019-0.0125	0.0054	0.0054	0.0052	0.0054
	difference (%)	-0.2	-4.6	-0.3
PND13 pups
Number of evaluated litters	11	9	9^§/10	11
T4 concentration (ng/mL) (NS) HC range: 26.3-61.6	44.11	42.80	43.66	42.05
	difference (%)	-3.0	-1.0	-4.7
Thyroid gland weights (g) (NS) HC range: 0.002-0.011	0.0054	0.0052	0.0050	0.0053
	difference (%)	-2.6	-6.8	-0.8
Thyroid gland / body weight (%) (NS) HC range: 0.96-3.63	1.8211	1.7202	1.6718	1.8073
	difference (%)	-5.5	-8.2	-0.8

Notes: Data (group mean values) were rounded to two or four decimal places

Thyroid and parathyroid weights were measured together. Thyroid gland weight for one male and one female pup per litter were determined except of litters #1511, #2508 and #2509 where no pups survived until PND13, furthermore four females (#2503, #3505, #3507 and #4502) had no litters.

Pups blood (plasma) was pooled for T4 (thyroxin) determination

HC: Historical control.

§Note: No thyroid hormone measurement was made for #3506 litter due to technical reason (no internal standard peak was seen)

NS: Statistically not significant when compared to control

Conclusions:

Based on the results observed in this combined repeated dose, reproductive/developmental toxicity screening study, the No Observed Adverse Effect Level (NOAEL) for 2-tert-butyl-1,4-dimethoxybenzene was determined to be 150 mg/Kg bw/day for systemic toxicity of the male parental (adult) animals (based on body weight and weight gain effects) and 450 mg/Kg bw/day for systemic toxicity of the female parental (adult) animals. The NOAEL for reproductive and developmental toxicity in rats was determined to be 450 mg/Kg bw/day, based on lack of adverse treatment-related effects observed at the highest dose tested.

Executive summary:

In a key Guideline OECD 422 combined repeated dose, reproductive/developmental toxicity screening study, the toxicity potential of the test material (2-tert-butyl-1,4-dimethoxybenzene; EC# 244-216-5) was evaluated in rats following repeated exposure via oral gavage. Crl:WI (Wistar) rats (12/sex/dose) were exposed to the test material via gavage once daily at doses of 0, 50, 150, or 450 mg/Kg bw/day for 2 weeks before mating, during mating, up to and including the day before the necropsy. The total exposure was 28 days in total for males while females were treated throughout gestation up to and including postpartum/lactation day (PPD) 13. The control group received the vehicle Polyethylene glycol 400 (PEG 400) only.

 

Parameters measured during the study included signs of morbidity and mortality, observation of clinical signs daily (general) or weekly (detailed), neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of locomotor activity was performed during the last week of treatment. Additionally, reproductive performance, pregnancy, parturition, and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND 13.

 

At termination, gross necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues / organs sampled and preserved in appropriate fixatives from the adult animals as well as offspring. Thyroxine (T4) levels in the adult males and PND 13 pups were also assessed. For the adult animals, a detailed histological examination was performed on a select list ofretained organs of 5 control and high dose animals per sex, all animals found dead, and all those male / female mating pairs where no conceptus was achieved. Based on the results of the histopathology evaluation of the control and high dose female rats, additional evaluation of liver and thymus samples were made using the remaining samples of the control and high dose groups, and all female animals of the mid dose and low dose groups.

 

No treatment-related mortality or clinical signs of toxicity were observed through the study period. Treatment-related reduced body weight gain was observed in high dose male rats (with weight loss on the first week) but no such effect on body weight or body weight gain was observed in the female animals through the study period. Food consumption remained unaffected by treatment.

 

Haematology, coagulation, and clinical chemistry parameters were not adversely impacted by exposure to the test material but discoloured (brown) urine was observed in the high and mid dose males at termination. However, this was not considered to be an adverse treatment-related effect.

 

Gross necropsy did not reveal any remarkable findings in any of the dose groups in both sexes.

 

A treatment-related effect on the liver weight of male and female rats was observed in the high and mid dose groups. The increased liver weight correlated with microscopic findings (minimal centrilobular hepatocellular hypertrophy) in 9/11 high dose group females. This histopathology finding was considered to be an adaptive, non-adverse change. A similar effect was not observed in livers of low dose group animals.

 

No treatment-related effect on neurotoxicity parameters was observed and the oestrus cycle of parental females was also unaffected by exposure to the test material. No treatment-related effects were noted in the reproductive parameters, gestation, parturition and lactation. There were no effects on the F1 offspring viability, clinical signs, and physical or sexual development. No treatment-related macroscopic findings were observed in F1 pups at necropsy.

 

No effect of the test material was observed in the study based on the results of thyroid hormone analysis. Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups. No treatment-related effect was observed on anogenital distance and nipple retention during the study. No nipples/areolae were counted for any male pups on PND 13. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, no endocrine signal was detected.

 

Based on the results observed in this combined repeated dose, reproductive/developmental toxicity screening study, the No Observed Adverse Effect Level (NOAEL) for 2-tert-butyl-1,4-dimethoxybenzene was determined to be 150 mg/Kg bw/day for systemic toxicity of the male parental (adult) animals (based on body weight and weight gain effects) and 450 mg/Kg bw/day for systemic toxicity of the female parental (adult) animals. The NOAEL for reproductive and developmental toxicity in rats was determined to be 450 mg/Kg bw/day, based on lack of adverse treatment-related effects observed at the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline OECD 422 study conducted in rats

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a key Guideline OECD 422 combined repeated dose, reproductive/developmental toxicity screening study (Charles River Laboratories Hungary Kft. (formerly Citoxlab Hungary Ltd.), 2020a), the toxicity potential of the test material (2-tert-butyl-1,4-dimethoxybenzene; EC# 244-216-5) was evaluated in rats following repeated exposure via oral gavage. Crl:WI (Wistar) rats (12/sex/dose) were exposed to the test material via gavage once daily at doses of 0, 50, 150, or 450 mg/Kg bw/day for 2 weeks before mating, during mating, up to and including the day before the necropsy. The total exposure was 28 days in total for males while females were treated throughout gestation up to and including postpartum/lactation day (PPD) 13. The control group received the vehicle Polyethylene glycol 400 (PEG 400) only.

 

Parameters measured during the study included signs of morbidity and mortality, observation of clinical signs daily (general) or weekly (detailed), neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of locomotor activity was performed during the last week of treatment. Additionally, reproductive performance, pregnancy, parturition, and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND 13.

 

At termination, gross necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues / organs sampled and preserved in appropriate fixatives from the adult animals as well as offspring. Thyroxine (T4) levels in the adult males and PND 13 pups were also assessed. For the adult animals, a detailed histological examination was performed on a select list ofretained organs of 5 control and high dose animals per sex, all animals found dead, and all those male / female mating pairs where no conceptus was achieved. Based on the results of the histopathology evaluation of the control and high dose female rats, additional evaluation of liver and thymus samples were made using the remaining samples of the control and high dose groups, and all female animals of the mid dose and low dose groups.

 

No treatment-related mortality or clinical signs of toxicity were observed through the study period. Treatment-related reduced body weight gain was observed in high dose male rats (with weight loss on the first week) but no such effect on body weight or body weight gain was observed in the female animals through the study period. Food consumption remained unaffected by treatment.

 

Haematology, coagulation, and clinical chemistry parameters were not adversely impacted by exposure to the test material but discoloured (brown) urine was observed in the high and mid dose males at termination. However, this was not considered to be an adverse treatment-related effect.

 

Gross necropsy did not reveal any remarkable findings in any of the dose groups in both sexes.

 

A treatment-related effect on the liver weight of male and female rats was observed in the high and mid dose groups. The increased liver weight correlated with microscopic findings (minimal centrilobular hepatocellular hypertrophy) in 9/11 high dose group females. This histopathology finding was considered to be an adaptive, non-adverse change. A similar effect was not observed in livers of low dose group animals.

 

No treatment-related effect on neurotoxicity parameters was observed and the oestrus cycle of parental females was also unaffected by exposure to the test material. No treatment-related effects were noted in the reproductive parameters, gestation, parturition and lactation. There were no effects on the F1 offspring viability, clinical signs, and physical or sexual development. No treatment-related macroscopic findings were observed in F1 pups at necropsy.

 

No effect of the test material was observed in the study based on the results of thyroid hormone analysis. Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups. No treatment-related effect was observed on anogenital distance and nipple retention during the study. No nipples/areolae were counted for any male pups on PND 13. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, no endocrine signal was detected.

 

Based on the results observed in this combined repeated dose, reproductive/developmental toxicity screening study, the No Observed Adverse Effect Level (NOAEL) for 2-tert-butyl-1,4-dimethoxybenzene was determined to be 150 mg/Kg bw/day for systemic toxicity of the male parental (adult) animals (based on body weight and weight gain effects) and 450 mg/Kg bw/day for systemic toxicity of the female parental (adult) animals. The NOAEL for reproductive and developmental toxicity in rats was determined to be 450 mg/Kg bw/day, based on lack of adverse treatment-related effects observed at the highest dose tested.

A supporting dose-range finding study (Charles River Laboratories Hungary Kft. (formerly Citoxlab Hungary Ltd.), 2020b) was conducted to evaluate the toxicity potential of the test material (2-tert-butyl-1,4-dimethoxybenzene) when administered daily via oral gavage to Wistar rats (4/sex/dose) at dose levels of 100, 300, or 1000 mg/Kg bw/day for a period of 7 days in order to determine the dose levels for a subsequent OECD Guideline 422 screening reproductive / developmental toxicity study. Control animals were treated with vehicle (PEG 400) only.

 

Mortality and clinical observations were observed twice daily. Body weight and food consumption were measured for all animals on Days 0, 3 and 6, and terminal body weight (fasted) was recorded at scheduled necropsy on Day 7. Following the daily repeated dose administration for 7 days, blood samples were collected for clinical pathology at necropsy from the surviving animals. Gross necropsy was performed at termination in each animal and selected tissues and organs were weighed and preserved in fixative.

 

No mortality or clinical signs of toxicity were observed through the study period. Body weight and body weight gain remained unaffected by treatment and no adverse treatment-related effects were observed on food consumption.

 

Treatment-related effects on haemoglobin concentration and haematocrit values were observed in the high- and mid-dose animals. Cell haemoglobin in high-dose female rats and platelet count in the high-dose male rats were also affected by treatment. Haematology parameters in the low-dose group animals were unaffected by treatment. The findings indicate a minor anaemia effect, which was considered to be adverse and treatment-related.

 

Treatment-related increases were observed in total protein and albumin concentrations, glucose concentration in animals of both sexes in the high-dose group while cholesterol values were affected in high- and mid-dose female rats. These changes indicate hepatic effects related to exposure to the test material.

 

Significantly higher liver weights observed in the high- (approx. 60%) and mid-dose (approx. 22%) groups were considered to be treatment-related. No histopathology was performed to confirm the liver weight effect as being treatment related or if the change was adverse or not. Gross necropsy did not reveal any remarkable findings.

 

Based on a significant increase in liver size, and minor changes to haematological and clinical chemistry parameters observed in the study, 1000 mg/Kg bw/day is not considered to be a suitable Maximum Tolerable Dose for the High dose level for the OECD 422 study in rats. Given the indications of changes at 100 mg/Kg bw/day after 7 days treatment (which may or may not become more pronounced with a longer treatment period), a low dose below 100 mg/Kg bw/day may be advisable.