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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

OECD 422 Combined Repeat Dose Toxicity study with Reproduction/Developmental Toxicity screening test:


The NOAEL for reproductive/developmental toxicity was 150 mg/kg/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:
25 September 2019 - 17 January 2020
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)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
- Intended use: Industrial chemical.
- Appearance: Yellowish, clear oil
- Storage conditions: Refrigerated (2 to 8°C), under argon and protected from light. Containers were kept sealed and flushed with argon after every use. Prolonged exposure to air and light was avoided.
- Supplier: Sponsor
- Batch number: 1394901004
- Expiry date: 1 January 2020
- Purity: >99% (by HPLC area % 225 nm bandwidth 50 nm)
Species:
rat
Strain:
Wistar
Remarks:
Han Wistar (RccHan™; WIST)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Envigo RMS Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males 84 to 90 days old; Females 98 to 104 days old.
- Weight at study initiation: Males 308 to 341 g; Females 193 to 242 g.
- Housing:
* Cages- 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.
* Bedding- Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
* Aspen chew block- A soft white untreated wood block; provided to each cage throughout the study (except during lactation) and replaced when necessary.
* Plastic shelter- Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
* Paper shavings- Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes; ad libitum.
- Acclimation period: Males six days before commencement of treatment; Females 20 days before commencement of treatment.

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. Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
ROUTE OF ADMINSTRATION
- Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
- Treated at: Constant doses in mg/kg/day.
- Volume dose: 5 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Frequency: Once daily at approximately the same time each day.

PREPARATION OF DOSING SOLUTIONS:
Formulation procedures were performed under yellow light. Starting with the lowest concentration, the required amount of test item was weighed and then mixed with the vehicle (approximately 50% of the final volume). The mixture was magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

PREPARATION
- Rate of preparation of diet (frequency): Weekly.
- Storage of formulation: Ambient temperature (15 to 25°C).
Details on mating procedure:
- Pairing commenced: After a minimum of two weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: The suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
- Achieved concentration: Samples of each of the first and last preparation formulations were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation).
The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
17 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected in consultation with the Sponsor based on the results of a 14-day preliminary toxicity study in the Han Wistar rat (Covance Study No. XY88WS). In that study, a dose level of 300 mg/kg/day was not tolerated, resulting in adverse signs including swaying, flattened gait, splayed hind limbs, reduced activity and piloerection. Macroscopically, lesions were identified in the stomach of the majority of the animals.
At 75 or 150 mg/kg/day, signs were generally limited to the first three days of treatment and consisted of piloerection, decreased activity and partially closed eyelids, with a few isolated incidences throughout the 14-day treatment period. Body weight gain was initially reduced but improved from Day 4, and food intake was slightly lower than the pretreatment values throughout treatment. After 14 days, liver and kidney weights were higher in males given 150 mg/kg/day and spleen weights were higher in females given 150 mg/kg/day, but there were no macroscopic findings.
It was therefore considered that 150 mg/kg/day was a suitable high dose for this study. The lower doses of 17 and 50 mg/kg/day (approximate factor of three between doses) were chosen in order to assess dose responsiveness of any effects and to obtain a clear no-observed-adverse-effect level (NOAEL).

- Rationale for animal assignment: weight of animals
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes.
Mortality check was performed near the start and end of each working day.

CLINICAL OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). 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.

BODY WEIGHT: Yes
F0 males:
Before dosing on the day that treatment commenced (Day 1) and weekly thereafter
On the day of necropsy.

F0 females:
Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption:
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3) but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 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: Yes / No / Not specified

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation. No significant effect was observed, and consequently quantitative measurements were not performed.

HAEMATOLOGY: Yes; At termination
- Anaesthetic used for blood collection: Yes, light general anesthesia induced by isoflurane
- Animals fasted: Not specified
- How many animals:
The five lowest numbered surviving males per group; The first five lactating females with a surviving litter per group
- Parameters checked:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

CLINICAL CHEMISTRY: Yes; At termination
- Anaesthetic used for blood collection: Yes, light general anesthesia induced by isoflurane
- Animals fasted: Not specified
- How many animals:
The five lowest numbered surviving males per group; The first five lactating females with a surviving litter per group
- Parameters checked:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Bile acids (Bi Ac)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Triglycerides (Trig)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)


Thyroid Hormone Analysis
- Blood samples were obtained as follows:
*At termination: All surviving F0 males; All surviving F0 females (no samples were obtained from animals which failed to litter or with total litter loss).
*Day 4 of age: Offspring: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation):
• one for T4 (serum)#
• one for TSH (serum)
*Day 13 of age: F1 offspring, two males and two females per litter (where possible)
• two for T4 (serum); where possible one male and one female#
• two for TSH (serum); where possible one male and one female
# Priority given to T4 sample.

- Conditions: No overnight deprivation of food.
- Anesthetic: Adults: Isoflurane; Offspring: None.
- Blood sample site: Adults: Sublingual vein; Offspring: Decapitation.
- Parameters: Thyroid stimulating hormone (TSH) and Thyroxine (T4)
- Anticoagulant: None.
- Blood volume: Adults- 1.0 mL; Offspring- max possible.
- Blood tubes: Greiner Minicollect tubes with clotting activator.
- Processing: Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
- Centrifugation conditions: At 2000g for ten minutes at 4°C.
- Aliquot volumes:
•Adults:
Aliquot 1: T4 - 0.2 mL of serum
Aliquot 2: TSH - residual serum
•Offspring:
T4 - 0.2 mL of serum
TSH - residual serum
- Final storage conditions: Deep frozen (approximately -60°C to -90°C), pending analysis.
Oestrous cyclicity (parental animals):
ESTROUS CYCLE
- Dry smears: For 15 days before pairing using cotton swabs.
- Wet smears: Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination.

PARTURITION OBSERVATIONS AND GESTATION LENGTH
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded, and any difficulties observed were recorded.
Litter observations:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
- Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
- Individual offspring body weights: Days 1, 4, 7 and 13 of age.
- Ano-genital distance: Day 1 - all F1 offspring.
- Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
SACRIFICE: All adult animals: Carbon dioxide asphyxiation (no exposure to carbon dioxide took place until after the completion of blood sampling for clinical pathology and thyroid hormone assays).

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

- Time of Necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 13 of lactation.
Postmortem examinations (offspring):
SACRIFICE:
Selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

- Time of Necropsy
F1 offspring Day 13 of age.
Statistics:
All statistical analyses were carried out separately for males and females.
Data relating to food consumption 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 sequence of statistical tests was used for implantations, litter size, sex ratio - percentage male, post implantation survival index, ano-genital distance data: Bartlett's test for variance homogeneity (Bartlett, 1937), Williams’ test (Williams, 1971; 1972), Dunnett's test (Dunnett, 1955; 1964) , Kruskal-Wallis’ test (Kruskal and Wallis, 1952; 1953), Wilcoxon rank sum tests (Wilcoxon, 1945), Shirley's test (Shirley, 1977), Steel's test (Steel, 1959), Fisher’s exact tests (Fisher, 1973), Cochran Armitage test (Armitage, 1955), Chi-square test, (Cytel, 1995).
Clinical signs:
no effects observed
Description (incidence and severity):
After dosing on Day 1, all animals receiving 150 mg/kg/day displayed signed of decreased activity and partially closed eyelids. No similar signs were recorded on any other occasion. Transient chin rubbing and salivation were recorded in animals receiving 150 mg/kg/day. These signs are commonly observed in studies where the test item is administered by oral gavage and, consequently, are considered of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
• A control male was found dead on Day 13 with no prior signs recorded.
• A female No in 150 mg/kg/day was killed for welfare reasons on Day 21 of gestation due to general skin pallor, a hunched posture, ungroomed coat and chromodacryorrhea.
• Two females were killed prematurely due to total litter loss (one in control and in 150 mg/kg/day group).
• Two females failed to litter and were therefore prematurely sacrificed on Day 25 of gestation (one control and one in 50 mg/kg/day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: Overall body weight gain (Day 1 to 36) was statistically significantly low, when compared with controls, in males receiving 150 mg/kg/day (71% of control).
Females: effect on body weight gain was less apparent; before pairing, body weight gain was lower than controls in females receiving 50 or 150 mg/kg/day but without dose response. During gestation and lactation, body weight gain was higher than controls at all BN-02 dose levels, but again there was no dose response.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean food intake was lower than control in males and in females before pairing at 150 mg/kg/day (93 and 92% of control in males and females, respectively), which was attributed to low food consumption during the first week of treatment. Food intake was unaffected in females during gestation or lactation.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination performed after 5 weeks of treatment for males and on Day 13 of lactation for females revealed no toxicologically significant differences from controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma performed revealed a treatment-related trend towards reduced plasma glucose concentration in males receiving 17, 50 or 150 mg/kg/day and in females receiving 150 mg/kg/day, with the extent of the difference from control being more apparent in males. There was also a reduction of creatinine concentration in males receiving 150 mg/kg/day, which was not present in females. Phosphorus concentration was statistically significant lower than control and potassium concentration was statistically significantly higher than control in females receiving 150 mg/kg/day. These findings were not apparent in males. All other differences from control were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Sensory Reactivity Observations and Grip Strength: Sensory reactivity and grip strength were unaffected by treatment.
- Motor Activity: There was no effect of treatment upon motor activity. All differences from control were transient and/or without dose relationship, and were considered unrelated to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings related to the treatment with the test substance administrated by oral gavage were seen in Han Wistar rats. The incidence and distribution of all findings were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No findings related to the treatment with the test substance administrated by oral gavage were seen in Han Wistar rats. The incidence and distribution of all findings were considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid Hormone Analysis
Serum thyroxine (T4) concentrations in adult males and offspring on Day 13 of age were considered to be unaffected by treatment.
In female offspring of parental animals receiving 150 mg/kg/day, serum T4 concentration was statistically significantly lower than control but there was no similar finding in male offspring. Conversely, in adult males receiving 150 mg/kg/day, T4 concentration was higher than control. Given the difference in the direction of the variation from control between the adults and offspring, the lack of a dose relationship and the lack of findings in male offspring, these findings were attributed to normal biological variation. As there was considered to be no effect of treatment, no further analysis of T4 or TSH was required.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval, gestation length and index, mating performance and fertility were considered unaffected by treatment with the test substance. All females showed diestrus at termination.
The oral administration of the test substance to rats at dose levels of 17, 50 or 150 mg/kg/day was generally well tolerated. There was no adverse effect of treatment upon the clinical condition of the animals, their behavior in the arena, sensory reactivity or grip strength. A reduction of body weight gain and food intake seen at 150 mg/kg/day in males throughout treatment, and also in females before pairing was considered to be a non-specific indicator of toxicity. This effect was considered adverse in males only.

Five premature deaths occurred during the study, however as three of these occurred in the control group, they were unrelated to treatment. After dosing on Day 1, all animals receiving 150 mg/kg/day displayed signs of decreased activity and partially closed eyelids. No similar signs were recorded on any other occasion, therefore this single, transient occurrence was considered non-adverse.

There were no histopathological correlates for the high liver weights, or any findings in the kidney to account for the reduced plasma glucose and creatinine concentrations or the variations to plasma and potassium, which were therefore considered non-adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs.
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was a trend towards lower overall body weight gain (Day 1-13) in the offspring of parental animals receiving 17, 50 or 150 mg/kg/day (91, 91 and 86% of control, respectively), which may be related to the slightly higher litter sizes in the treated groups.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The ano-gential distances of offspring were unaffected by parental treatment with the test substance.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The examination of offspring on Day 13 of age revealed no treatment-related findings.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination of offspring on Day 13 of age revealed no treatment-related findings.
Other effects:
no effects observed
Description (incidence and severity):
Litter Size, Sex Ratio and Survival Indices
Litter size, offspring survival to Day 13 of age and sex ratio were not adversely affected by parental treatment with the test substance.
The numbers of implantations and live offspring were generally higher than controls in females receiving the test substance at 17, 50 or 150 mg/kg/day.
There were no treatment related clinical signs, and ano-gential distances, litter size, offspring survival to Day 13 of age, and sex ratio were not adversely affected by parental treatment with the test substance.
Offspring body weight gain was slightly low in the offspring of parental animals receiving 17, 50 or 150 mg/kg/day which may be related to the slightly higher litter sizes in the treated groups.
The macroscopic examination of offspring on Day 13 of age revealed no treatment-related findings.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes

The oral administration of the test substance to rats at dose levels of 17, 50 or 150 mg/kg/day was generally well tolerated. There was no adverse effect of treatment upon the clinical condition of the animals, their behavior in the arena, sensory reactivity or grip strength. A reduction of body weight gain and food intake seen at 150 mg/kg/day in males throughout treatment, and also in females before pairing was considered to be a non-specific indicator of toxicity. This effect was considered adverse in males only.

Five premature deaths occurred during the study, however they were unrelated to treatment.

This study included a screen for reproductive/developmental effects and the results obtained were unremarkable. Estrous cycles, pre-coital interval, gestation length and index, mating performance, fertility, litter size, offspring survival to Day 13 of age and sex ratio were not adversely affected by treatment. The decreased offspring body weight gain reported in the litters of parental animals receiving 17, 50 or 150 mg/kg/day was likely to be related to the slightly higher litter sizes in the treated groups and was non-adverse.

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone thyroxine (T4) in adult males and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and, because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. There was a decrease of circulating levels of thyroxine in in female offspring of parental animals receiving 150 mg/kg/day, and conversely, there was an increase in adult males receiving 150 mg/kg/day. There was however, no dose-relationship and no constancy with regard to the direction of the variation from control. In addition, there were no significant changes to the weight of the thyroid gland at necropsy, or any histopathological findings in the thyroid gland indicative of a disruption to T4 production (such as follicular cell hypertrophy). These findings were there attributed to normal biological variation, and not to treatment. No treatment-related histopathological findings were identified in the reproductive organs of any animal. All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distances and male nipple counts were not adversely affected by treatment.  It was therefore concluded that, in the context of this study, the test substance showed no evidence of being an endocrine disruptor.   

There were no histopathological correlates for the high liver weights, or any findings in the kidney to account for the reduced plasma glucose and creatinine concentrations or the variations to plasma and potassium, which were therefore considered non-adverse.

Conclusions:
It is concluded that the oral administration of the test substance to Han Wistar rats at dose levels of 17, 50 or 150 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was generally tolerated in the adult animals. Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, the substance showed no evidence of being an endocrine disruptor.

The NOAEL for reproductive/developmental toxicity was 150 mg/kg/day.
Executive summary:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted (Covance Laboratories Limited, 2020, Study TP20CQ) to assess the general toxicity and reproductive toxicity of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine in the Han Wistar rat. The study was conducted in accordance with OECD 422 guideline and in compliance with GLP.

Three groups of ten male and ten female rats received the test substance at doses of 17, 50 or 150 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

The oral administration of the test substance to rats was generally well tolerated. There was no adverse effect of treatment upon the clinical condition of the animals, their behavior in the arena, sensory reactivity or grip strength. A reduction of body weight gain and food intake seen at 150 mg/kg/day in males throughout treatment, and also in females before pairing was considered to be a non-specific indicator of toxicity. This effect was considered adverse in males only. Five premature deaths occurred during the study they were unrelated to treatment. After dosing on Day 1, all animals receiving 150 mg/kg/day displayed signs of decreased activity and partially closed eyelids. No similar signs were recorded on any other occasion, therefore this single, transient occurrence was considered non-adverse.

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, the test substance showed no evidence of being an endocrine disruptor. The decreased offspring body weight gain reported in the litters of parental animals receiving 17, 50 or 150 mg/kg/day was likely to be related to the slightly higher litter sizes in the treated groups and was non-adverse.

The study design also included measurement of the hormone thyroxine (T4) in adult males and in offspring at Day 13 of age. Changes in adult organ weight and gross organ pathology of endocrine-sensitive organs and an external examination of all offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age were evaluated. There was a decrease of circulating levels of thyroxine in in female offspring of parental animals receiving 150 mg/kg/day, and there was an increase in adult males receiving 150 mg/kg/day. There was no dose-relationship and no constancy with regard to the direction of the variation from control. In addition, there were no significant changes to the weight of the thyroid gland at necropsy, or any histopathological findings in the thyroid gland indicative of a disruption to T4 production (such as follicular cell hypertrophy). All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distances and male nipple counts were not adversely affected by treatment.  It was therefore concluded that, in the context of this study, the test substance showed no evidence of being an endocrine disruptor. 

  

There were no histopathological correlates for the high liver weights, or any findings in the kidney to account for the reduced plasma glucose and creatinine concentrations or the variations to plasma and potassium, which were therefore considered non-adverse.

Due to the adverse effect on body weight gain in males at 150 mg/kg/day, the no‑observed‑adverse‑effect level (NOAEL) of the substance for systemic toxicity was considered to be 50 mg/kg/day for males and 150 mg/kg/day for females. The NOAEL for reproductive/developmental toxicity was 150 mg/kg/day.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant guideline 'Combined Repeat Dose Toxicity study with Reproduction/Developmental screening test' with a klimisch score of 1
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 GLP compliant, sub-acute OECD 422 guideline 'Combined Repeat Dose Toxicity study with Reproduction/Developmental screening test' it is concluded that the oral administration of the test substance to Han Wistar rats at dose levels of 17, 50 or 150 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was generally tolerated in the adult animals. Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, the substance showed no evidence of being an endocrine disruptor. The NOAEL for reproductive/developmental toxicity was 150 mg/kg/day.

Effects on developmental toxicity

Description of key information

OECD 422 Combined Repeat Dose Toxicity study with Reproduction/Developmental Toxicity screening test:


The NOAEL for reproductive/developmental toxicity was 150 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP compliant guideline 'Combined Repeat Dose Toxicity study with Reproduction/Developmental screening test' with a klimisch score of 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP compliant, sub-acute OECD 422 guideline 'Combined Repeat Dose Toxicity study with Reproduction/Developmental screening test it is concluded that the oral administration of the test substance to Han Wistar rats at dose levels of 17, 50 or 150 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was generally tolerated in the adult animals. Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, the substance showed no evidence of being an endocrine disruptor.    

The NOAEL for reproductive/developmental toxicity was 150 mg/kg/day.

Justification for classification or non-classification

GLP compliant guideline 'Combined Repeat Dose Toxicity study with Reproduction/Developmental screening test' with a klimisch score of 1

Additional information