Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Endpoint summary

Description of key information

Repeated dose toxicity, oral: NOAEL = 400 mg/kg bw/day (OECD 422 - oral gavage, GLP, Rel.1)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 October 2020 to 25 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: between 200 and 341 g at the initiation of dosing
- Fasting period before study: no
- Housing: On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dose level, and sex. Cages were arranged on the racks in group order.
- Enrichment: For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if necessary. Periodic analysis of the water is performed.
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-10-09 To: 2021-01-04

Route of administration:

oral: gavage

Details on route of administration:

The route of administration was oral (gavage) because this is a potential route of exposure to humans. Historically, this route has been used extensively for studies of this nature.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared approximately weekly, and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5°C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing.

VEHICLE
- Vehicle: Corn oil. The vehicle, corn oil, was dispensed approximately weekly for administration to Group 1 control animals and preparation of the test substance formulations.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light
- Supplier: Charles River Laboratories

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis performed by a gas chromatography method with flame ionization detection using a validated analytical procedure.
- Concentration and homogeneity analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with each individual sample concentration within ± 20% of the target concentration.
Homogeneity results were considered acceptable if the relative standard deviation of
the mean value at each sampling location was ≤ 10% and if mean sample concentration results
were within or equal to ± 15% of theoretical concentration.
- Stability and Resuspension Homogeneity Analysis: Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 8 days refrigerated (target of 5°C) (Akalkotkar, Draft, 00810034). Therefore, stability and resuspension homogeneity of test substance formulations were not assessed on this study.

Duration of treatment / exposure:

Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days.
Females were dosed for 14 days prior to mating, and continuing through 1 day prior to euthanasia.
The F1 animals were not directly exposed to the test substance at any time during the study; the
offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose. All animals were dosed at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (Group 1)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test item (Group 2)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Test item (Group 3)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Test item (Group 4)

No. of animals per sex per dose:

10 animals/sex/dose

RATIONALE FOR NUMBER OF ANIMALS
The number of animals was based on the OECD Guideline for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 July 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females were evaluated for estrous cyclicity during the pretest period, and any females that fail to exhibit normal 4–5 day estrous cycles (e.g., EDDDE) during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels were determined from results of previous studies (Calkins, Draft, 00810035). In a previous study, ethyl safranate was administered to male and female Sprague Dawley rats via oral gavage for 14 days at dose levels of 100, 300, 600, and 1000 mg/kg/day. In that study, all animals survived to the scheduled necropsy, and remarkable clinical observations were restricted to wet fur around the mouth in the 300, 600, and 1000 mg/kg/day males and 600 and 1000 mg/kg/day females. Lower mean body weight gains with corresponding lower food consumption were noted in the 1000 mg/kg/day males during Study Days 0–4. There were no effects on body weight or food consumption in females at any dose level. Changes in organ weights (absolute, relative to final body weight, and/or relative to brain weight) included higher liver weights for males and females at 300, 600, and 1000 mg/kg/day and lower thymus weights for females all dose levels. As such, dose levels of 100, 200, and 400 mg/kg/day were selected for this study.

SELECTION, ASSIGNMENT AND DISPOSITION OF ANIMALS
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals in poor health, at extremes of body weight range, or not exhibiting normal 4- to 5-day estrous
cycles were not assigned to groups.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital (following thyroid hormone blood collection for pups used for blood collection) and discarded.

Observations and examinations performed and frequency:

MORTALITY/VIABILITY: Yes
- Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were removed from the cage, and a detailed clinical observation was performed
once daily throughout the study. During the dosing period, these observations were performed
prior to dosing. On dosing days, clinical observations were also recorded 1 hour postdose. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured twice weekly until cohabitation. Once evidence
of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, and 13.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY, CLINICAL CHEMISTRY AND THYROID HORMONE ANALYSIS: Yes
- Clinical assessments were performed on 5/animals/sex/group at necropsy. Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from a jugular vein for males and from the retro-orbital sinus from animals anesthetized with isoflurane for females. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.
- Parameters checked in table 7.6.1/1 were examined.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- FOB assessments recorded for 5 animals/sex/group during the last week of dosing (Day 27; F0 males) or on Lactation Day 13 (F0 females). Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was performed in a sound-attenuated room equipped with a white noise generator. All animals were observed for the following parameters:
- Home Cage Observations: Posture/Body Carriage/Convulsions/Stereotypy/Tremor/Palpebral Closure/Ptosis
- Handling Observations: Ease of Removal/Handling Reactivity
- Open Field Observations: Rearing/Arousal/Alertness/Gait/Mobility/Vocalizations/Tremor/Respiration/Defecation/Stereotypy/Convulsions/Appearance/Lacrimation/Salivation/Exophthalmus/Palpebral Closure/Ptosis/Erected Fur
- Sensory Observations: Touch Response/Tactile Reflex/Startle Response/Tail Pinch Response/Pupil Response/Body Temperature
- Neuromuscular Observations: Body Tone/Grip strength-hind and forelimb/Rotarod performance/Hindlimb foot splay/Air Righting Reflex
- Physiological Observations: Catalepsy
- Motor activity was assessed for 5 animals/sex/group during the last week of dosing (Day 27; F0 males) or on Lactation Day 13 (F0 females). The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5-minute epochs over a period of 60 minutes, and the data were reported in 10-minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

IMMUNOLOGY: No

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

Sacrifice and pathology:

Terminal procedures were presented below in Table 7.6.1/2.

GROSS PATHOLOGY: Yes
- Unscheduled Deaths: No animals died during the course of the study.
- Scheduled Euthanasia: All surviving animals were euthanized by carbon dioxide inhalation.
- Necropsy: Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. Postimplantation loss was calculated for each female by subtracting the number of pups born from the number of implantation sites observed.

ORGAN WEIGHTS: Yes
- The following organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
- Organs Weighed at Necropsy: Adrenal glands/Brain/Epididymides/Heart/Kidneys/Liver/Ovaries with oviducts/Pituitary gland/Prostate gland/Seminal vesicle (with coagulating gland and fluid)/Spleen/Testes/Thymus gland/Thyroids with parathyroids (after fixation).
- Representative samples of the tissues identified in Table 7.6.1/3 were collected from all animals
and preserved in 10% neutral buffered formalin, unless otherwise indicated.

HISTOLOGY: Yes
- Tissues identified in Table 7.6.1/3 from 5 animals/sex in the control and high-dose groups, as well as gross lesions, liver (males and females), and kidneys (males only) from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.

HISTOPATHOLOGY: Yes
- Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues
identified in table 7.6.1/3 for microscopic examination were evaluated from 5 animals/sex in
the control and high-dose groups. Gross lesions, liver (males and females), and kidneys (males
only) were examined from all groups.

Optional endpoint(s):

Optional endpoints: No

Statistics:

Data collected during the predose period were not tabulated, summarized, or statistically analyzed, unless applicable to analyses in the proceeding sections. All statistical analyses were performed within the respective study phase, unless otherwise noted. Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as
indicated below according to sex and occasion or by litter. Values may also be expressed as a
percentage of pretreatment period or control values, or fold change of control values, when
deemed appropriate. Calculated values on Provantis tables may not be reproducible from the
individual values presented because all calculations were conducted using non-rounded values.

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were
conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise
noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses were performed according to the matrix attached in the full report study (p.35) when possible, but will exclude any group with less than 3 observations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related wet fur on the lower jaw and mouth were sporadically noted for 1-3 males and females in the 400 mg/kg/day group at approximately 1 hour postdosing. Wet fur around the mouth was also noted for females in the 200 mg/kg/day group at approximately 1 hour postdosing. These findings were considered non-adverse.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day group males were
unaffected by test substance administration throughout the study. None of the differences from
the control group were statistically significant.
FEMALES
*Twice weekly: Test substance-related higher mean body weight gains were noted in the 200 and 400 mg/kg/day group females during the premating period; differences were statistically significant when the entire premating period (Study Days 0-13) was evaluated. As a result, mean body weights in the 200 and 400 mg/kg/day groups that were 4.8% and 5.5% higher than the control group, respectively, on Study Day 13; differences were not statistically significant. Due to the minimal magnitude of difference from the control group, these effects were not considered adverse. Mean body weights and body weight gains in the 100 mg/kg/day group females was unaffected by test substance administration during the premating period.
*Gestation: Mean body weights and body weight gains in the 100, 200, and 400 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.
*Lactation: Test substance-related higher mean body weight gains were noted in the 400 mg/kg/day group females during lactation. The difference was statistically significant when the entire lactation period (Lactation Days 1-13) was evaluated. As a result, mean body weights in the
400 mg/kg/day group was 4.6% higher than the control group on Lactation Day 13; the difference was not statistically significant. Due to the minimal magnitude of difference from the control group, these effects were not considered adverse. Mean body weights and body weight gains in the 100 and 200 mg/kg/day groups were unaffected by test substance administration during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
Mean food consumption, evaluated as g/animal/day, in the 100, 200, and 400 mg/kg/day group
males was similar to that in the control group throughout the study. No statistically significant
differences were observed.
FEMALES
*Twice weekly: Mean food consumption in the 400 mg/kg/day group females was slightly higher (not statistically significant) than the control group throughout the premating period, corresponding to the higher body weight gains noted during this period. Mean food consumption in the 100 and 200 mg/kg/day group females was similar to that in the control group throughout the premating dosing period.
*Gestation: Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 200, 400 mg/kg/day groups was unaffected by test substance administration during gestation. Statistically significantly higher mean food consumption was note for females in the
400 mg/kg/day group during Gestation Days 4-7; however, this was transient. No other differences from the control group were statistically significant.
*Lactation: Mean food consumption in the 400 mg/kg/day group females was higher than the control group throughout the lactation period, corresponding to the higher body weight gains noted during this period; differences were generally statistically significant. Mean food consumption in the 100 and 200 mg/kg/day group females was similar to that in the control group during lactation.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Hematology: Females in the 200 and 400 mg/kg/day groups were noted with higher mean absolute monocyte levels and in the 100, 200, and 400 mg/kg/day group were noted with test substance-related higher mean absolute lymphocyte levels, relative to the control group. Differences in mean lymphocyte levels at 400 mg/kg/day were statistically significant. No other test substance-related effects were noted on hematology parameters at any dosage level.
- Coagulation: Males in the 400 mg/kg/day group were noted with test substance-related statistically significantly higher mean fibrinogen levels. Mean prothrombin time (PT) in 200 and
400 mg/kg/day group males and females were statistically significantly lower than the control
group. However, these values were within the range of values in the historical control data, and
as such the noted differences were not considered test substance-related. Moreover, decreased PT is not considered toxicologically relevant. No other test substance-related effects were noted on coagulation parameters at any dosage level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects for males in the 400 mg/kg/day group included statistically significantly higher mean cholesterol, total protein, and albumin and statistically significantly lower mean chloride. In addition, for females, test substance-related lower mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values were noted at all dosage levels and lower alkaline phosphatase (ALP) values were noted at 200 and 400 mg/kg/day groups; differences in ALT were statistically significant. No other test substance-related effects were noted on serum chemistry parameters. Statistically significantly lower mean chloride was noted for males in the 100 mg/kg/day group; however, this did not occur in a dose-related manner. Other differences from the control group were slight and not statistically significant.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher mean TSH levels with corresponding slightly higher T3 levels were noted in males at 400 mg/kg/day relative to the control group; differences in TSH were statistically significant. Due to a lack of corresponding effects on thyroid weights or microscopic correlates, the effects on TSH and T3 were considered non-adverse. T3, T4, and TSH levels at 100 and 200 mg/kg/day were generally similar to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Neurobehavioral assessments: Neurobehavioral assessments were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Day 27 (males) or on Lactation Day 13 (females).
- Motor activity: Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Day 27 (males) and Lactation Day 13 (females). Values obtained from the 6 sub-intervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated at Study Day 27 (males) and Lactation Day 13 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher mean kidney weights (absolute and relative to terminal body and
brain weight) were observed in the 400 mg/kg/day group males and females. Higher mean kidney weights were dose-responsive in the females (i.e., absolute, and relative to terminal brain weight). There were no microscopic correlates.

Test substance-related higher mean liver weights were identified in the 200 mg/kg/day group females and 400 mg/kg/day group males and females. Higher mean liver weights (absolute and
relative to terminal body and brain weight) were dose-responsive in the treated females, and correlated microscopically with minimal hepatocellular hypertrophy in the 400 mg/kg/day group
males and females.

No other test substance-related organ weight changes were noted. Lower mean prostate gland and thymus weights (absolute and relative to terminal body and brain weight) were observed in
the treated males when compared to control group males, but were considered incidental as mean and individual absolute values were within the normal range for the Charles River Ashland
historical control database and there were no microscopic correlates.

Remaining isolated organ weight values not discussed that were statistically different from their
respective controls exhibited no patterns, trends, or correlating data to suggest these values were
toxicologically relevant. Thus, other organ weight differences observed were considered incidental and unrelated to administration of ethyl safranate.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were
of similar incidence in control and treated animals and, therefore, were considered unrelated to
administration of ethyl safranate.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic changes identified in the kidneys of the 100, 200, and 400 mg/kg/day group males consisted of minimal hyaline droplet accumulation, single-cell tubular necrosis, tubular basophilia, and/or granular casts. Hyaline droplet accumulation (100, 200, and 400 mg/kg/day group males) was most prominent in the proximal convoluted tubules and was characterized as brightly eosinophilic, variably-sized intracytoplasmic droplets within tubular epithelial cells. Single cell-necrosis (400 mg/kg/day group males) was characterized by rare rounded, hypereosinophilic tubular epithelial cells within the lumen of the proximal convoluted tubules. Tubular basophilia (200 and 400 mg/kg/day group males) was characterized as basophilic tubules with prominent nuclei. Finally, tubular granular casts (400 mg/kg/day group males) were characterized as rare dilated tubules at the corticomedullary junction containing eosinophilic granular material. No test substance-related microscopic findings were identified in the kidneys in the treated females. The spectrum of renal findings in the treated males were suggestive of alpha-2u globulin nephropathy (Hard et al., 1993). Hyaline droplet accumulation composed of alpha-2u globulin is common in young control group male rats.
Formation and accumulation of hyaline droplets is accentuated by a wide range of xenobiotics termed CIGA (chemicals inducing alpha-2u globulin accumulation) through a mechanism of reversible, non-covalent binding to alpha-2u globulin that decreases the rate of degradation by proteolytic enzymes. Alpha-2u globulin accumulation is a male rat-specific effect, not occurring
in female rats or other species, including humans (Hamamura et al., 2006). Given the minimal
severity grade of microscopic findings and absence of clinical pathology alterations indicative of
renal dysfunction, these changes were considered non-adverse.
Test substance-related microscopic changes were present in the liver of the 400 mg/kg/day group males and females and consisted of minimal hepatocellular hypertrophy. Hepatocellular
hypertrophy was characterized as centrilobular to diffuse enlargement of hepatocytes.
Hepatocellular hypertrophy was considered nonadverse given the minimal severity grade and
absence of elevated liver enzymes.
Remaining microscopic findings not discussed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in
control and treated animals and, therefore, were considered unrelated to administration of ethyl
safranate.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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

Details on results:

The analyzed dosing formulations contained 98.8% to 112% of the test substance, which was
within the protocol-specified range of target concentrations for suspensions (85% to 115%), and
were homogeneous. The test substance was not detected in the analyzed vehicle formulation that
was administered to the control group (Group 1).

Females in the 200 and 400 mg/kg/day group were noted with higher mean absolute monocyte
levels and in the 100, 200, and 400 mg/kg/day groups were noted with test substance-related
higher mean absolute lymphocyte levels, relative to the control group. Males in the 400 mg/kg/day group were noted with test substance-related higher mean fibrinogen levels. No other test substance-related effects were noted on hematology or coagulation parameters at any
dosage level.

Test substance-related serum chemistry effects for males in the 400 mg/kg/day group included
higher mean cholesterol, total protein, and albumin and lower mean chloride. In addition, test
substance-related lower mean alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) values were noted for females at all dosage levels and lower alkaline phosphatase (ALP)
values were noted for females in the 200 and 400 mg/kg/day groups. These findings were
considered nonadverse. No other test substance-related effects were noted on serum chemistry
parameters.

Test substance-related higher mean TSH levels with corresponding slightly higher T3 levels were noted in males at 400 mg/kg/day relative to the control group. Due to a lack of corresponding effects on thyroid weights or microscopic correlates, the effects on TSH and T3 were considered non-adverse.

Test substance-related higher mean kidney weights were observed in the 400 mg/kg/day group
males and females. Higher mean kidney weights (absolute and relative to terminal body and
brain weight) were dose-responsive in the females. There were no microscopic correlates.

Test substance-related higher mean liver weights were identified in the 200 mg/kg/day group
females and 400 mg/kg/day group males and females. Higher mean liver weights (absolute and
relative to terminal body and brain weight) were dose-responsive in the treated females, and
correlated microscopically with minimal hepatocellular hypertrophy in the 400 mg/kg/day group
males and females. Hepatocellular hypertrophy was considered non-adverse given the minimal
severity grade and absence of elevated liver enzyme levels.

Test substance-related microscopic changes identified in the kidneys of males consisted of
minimal hyaline droplet accumulation (100, 200, and 400 mg/kg/day group males), single-cell
tubular necrosis (400 mg/kg/day group males), tubular basophilia (200 and 400 mg/kg/day group males), and granular casts (400 mg/kg/day group males). Given the minimal severity grade of microscopic findings and absence of clinical pathology alterations indicative of renal dysfunction, these changes were considered non-adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of this screening study, due to the absence of any adverse effects at any dosage level, a dosage level of 400 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive and systemic toxicity of ethyl safranate when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in compliance with GLP to evaluate the potential toxic effects of ethyl safranate when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.

 

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13.

The study design was as follows:

Group	Treatment	Dosage Level (mg/kg/day)^a	Dose concentration (mg/mL)	Dose Volume (mL/kg)	Number of animals
					Males	Females
1	Control	0	0	5	10	10
2	Ethyl Safranate	100	20	5	10	10
3	Ethyl Safranate	200	40	5	10	10
4	Ethyl Safranate	400	80	5	10	10

^aNot corrected for salt, purity, and water content.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, locomotor activity, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations. 

 

Results

The analyzed dosing formulations contained 98.8% to 112% of the test substance, which was within the protocol-specified range of target concentrations for suspensions (85% to 115%), and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

No test substance-related effects were noted on F0 survival, neurobehavior, motor activity, or gross macroscopic findings. 
All animals survived to the scheduled necropsy. Test substance-related wet fur on the lower jaw and mouth were sporadically noted for 1-3 males and females in the 400 mg/kg/day group at approximately 1-hour postdosing. Wet fur around the mouth was also noted for females in the 200 mg/kg/day group at approximately 1-hour postdosing. These findings were considered non-adverse. No other test substance-related clinical findings were noted.

Test substance-related higher mean body weight gains were noted for females in the 200 and 400 mg/kg/day groups generally throughout the premating period (Study Days 0-13) and for females in the 400 mg/kg/day group generally throughout lactation (Lactation Days 1-13).
Corresponding higher mean food consumption was noted for females in the 400 mg/kg/day group during these periods. As a result, mean body weights for these females were 4.8% and 5.5% higher in the 200 and 400 mg/kg/day groups, respectively, on Study Day 13 and mean body weight in the 400 mg/kg/day group was 4.6% higher than the control group on Lactation Day 13. These findings were considered non-adverse. No other effects were noted on body weights, body weight gains, or food consumption.

Females in the 200 and 400 mg/kg/day group were noted with higher mean absolute monocyte levels and in the 100, 200, and 400 mg/kg/day groups were noted with test substance-related higher mean absolute lymphocyte levels, relative to the control group. Males in the 400 mg/kg/day group were noted with test substance-related higher mean fibrinogen levels. No other test substance-related effects were noted on hematology or coagulation parameters at any
dosage level.

Test substance-related serum chemistry effects for males in the 400 mg/kg/day group included higher mean cholesterol, total protein, and albumin and lower mean chloride. In addition, test substance-related lower mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values were noted for females at all dosage levels and lower alkaline phosphatase (ALP) values were noted for females in the 200 and 400 mg/kg/day groups. These findings were
considered non-adverse. No other test substance-related effects were noted on serum chemistry parameters.

Test substance-related higher mean TSH levels with corresponding slightly higher T3 levels were noted in males at 400 mg/kg/day relative to the control group, but were considered non-adverse due to a lack of corresponding effects on thyroid weights or microscopic correlates.

Test substance-related higher mean kidney weights were observed in the 400 mg/kg/day group males and females. Higher mean kidney weights (absolute and relative to terminal body and brain weight) were dose-responsive in the females. There were no microscopic correlates.

Test substance-related higher mean liver weights were identified in the 200 mg/kg/day group females and 400 mg/kg/day group males and females. Higher mean liver weights (absolute and relative to terminal body and brain weight) were dose-responsive in the treated females, and correlated microscopically with minimal hepatocellular hypertrophy in the 400 mg/kg/day group males and females. Hepatocellular hypertrophy was considered non-adverse given the minimal severity grade and absence of liver enzyme alterations.

Test substance-related microscopic changes identified in the kidneys of males consisted of minimal hyaline droplet accumulation (100, 200, and 400 mg/kg/day group males), single-cell tubular necrosis (400 mg/kg/day group males), tubular basophilia (200 and 400 mg/kg/day group males), and granular casts (400 mg/kg/day group males). Given the minimal severity grade of microscopic findings and absence of clinical pathology alterations indicative of renal dysfunction, these changes were considered non-adverse.

Additional paramaters and endpoints evaluated were available in IUCLID 7.8.1.

 

Under the conditions of this screening study, due to the absence of any adverse effects at any dosage level, a dosage level of 400 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive and systemic toxicity of ethyl safranate when administered orally by gavage to Crl:CD(SD) rats.

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the dose range-finding study for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Calkins, 2021), the test item was administered to male and female Sprague Dawley rats via oral gavage for 14 days at dose levels of 100, 300, 600, and 1000 mg/kg/day. In that study, all animals survived to the scheduled necropsy, and remarkable clinical observations were restricted to wet fur around the mouth in the 300, 600, and 1000 mg/kg/day males and 600 and 1000 mg/kg/day females. Lower mean body weight gains with corresponding lower food consumption were noted in the 1000 mg/kg/day males during Study Days 0–4. There were no effects on body weight or food consumption in females at any dose level. Changes in organ weights (absolute, relative to final body weight, and/or relative to brain weight) included higher liver weights for males and females at 300, 600, and 1000 mg/kg/day and lower thymus weights for females all dose levels. As such, dose levels of 100, 200, and 400 mg/kg/day were selected for the main study.

 

In the main study (OECD 422), considered as the key study (Calkins, 2021), the potential toxic effects of ethyl safranate was evaluated when administered to rats for 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.

 

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13.

The study design was as follows:

Group	Treatment	Dosage Level (mg/kg/day)^a	Dose concentration (mg/mL)	Dose Volume (mL/kg)	Number of animals
					Males	Females
1	Control	0	0	5	10	10
2	Ethyl Safranate	100	20	5	10	10
3	Ethyl Safranate	200	40	5	10	10
4	Ethyl Safranate	400	80	5	10	10

^aNot corrected for salt, purity, and water content.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, locomotor activity, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations. 

 

Results

The analyzed dosing formulations contained 98.8% to 112% of the test substance, which was within the protocol-specified range of target concentrations for suspensions (85% to 115%), and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

No test substance-related effects were noted on F0 survival, neurobehavior, motor activity, or gross macroscopic findings. 
All animals survived to the scheduled necropsy. Test substance-related wet fur on the lower jaw and mouth were sporadically noted for 1-3 males and females in the 400 mg/kg/day group at approximately 1-hour postdosing. Wet fur around the mouth was also noted for females in the 200 mg/kg/day group at approximately 1-hour postdosing. These findings were considered non-adverse. No other test substance-related clinical findings were noted.

Test substance-related higher mean body weight gains were noted for females in the 200 and 400 mg/kg/day groups generally throughout the premating period (Study Days 0-13) and for females in the 400 mg/kg/day group generally throughout lactation (Lactation Days 1-13).
Corresponding higher mean food consumption was noted for females in the 400 mg/kg/day group during these periods. As a result, mean body weights for these females were 4.8% and 5.5% higher in the 200 and 400 mg/kg/day groups, respectively, on Study Day 13 and mean body weight in the 400 mg/kg/day group was 4.6% higher than the control group on Lactation Day 13. These findings were considered non-adverse. No other effects were noted on body weights, body weight gains, or food consumption.

Females in the 200 and 400 mg/kg/day group were noted with higher mean absolute monocyte levels and in the 100, 200, and 400 mg/kg/day groups were noted with test substance-related higher mean absolute lymphocyte levels, relative to the control group. Males in the 400 mg/kg/day group were noted with test substance-related higher mean fibrinogen levels. No other test substance-related effects were noted on hematology or coagulation parameters at any
dosage level.

Test substance-related serum chemistry effects for males in the 400 mg/kg/day group included higher mean cholesterol, total protein, and albumin and lower mean chloride. In addition, test substance-related lower mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values were noted for females at all dosage levels and lower alkaline phosphatase (ALP) values were noted for females in the 200 and 400 mg/kg/day groups. These findings were
considered non-adverse. No other test substance-related effects were noted on serum chemistry parameters.

Test substance-related higher mean TSH levels with corresponding slightly higher T3 levels were noted in males at 400 mg/kg/day relative to the control group, but were considered non-adverse due to a lack of corresponding effects on thyroid weights or microscopic correlates.

Test substance-related higher mean kidney weights were observed in the 400 mg/kg/day group males and females. Higher mean kidney weights (absolute and relative to terminal body and brain weight) were dose-responsive in the females. There were no microscopic correlates.

Test substance-related higher mean liver weights were identified in the 200 mg/kg/day group females and 400 mg/kg/day group males and females. Higher mean liver weights (absolute and relative to terminal body and brain weight) were dose-responsive in the treated females, and correlated microscopically with minimal hepatocellular hypertrophy in the 400 mg/kg/day group males and females. Hepatocellular hypertrophy was considered non-adverse given the minimal severity grade and absence of liver enzyme alterations.

Test substance-related microscopic changes identified in the kidneys of males consisted of minimal hyaline droplet accumulation (100, 200, and 400 mg/kg/day group males), single-cell tubular necrosis (400 mg/kg/day group males), tubular basophilia (200 and 400 mg/kg/day group males), and granular casts (400 mg/kg/day group males). Given the minimal severity grade of microscopic findings and absence of clinical pathology alterations indicative of renal dysfunction, these changes were considered non-adverse.

Additional paramaters and endpoints evaluated were available in IUCLID 7.8.1.

 

Under the conditions of this screening study, due to the absence of any adverse effects at any dosage level, a dosage level of 400 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive and systemic toxicity of ethyl safranate when administered orally by gavage to Crl:CD(SD) rats.

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

 

Justification for classification or non-classification

 

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

 

Self-classification:

In a recent GLP combined repeated dose toxicity study with the reproduction / developmental screening test (OECD guideline 422), based on the absence of any adverse effects at any dosage level, a dosage level of 400 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive and systemic toxicity of ethyl safranate when administered orally by gavage to Crl:CD(SD) rats.

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