Ethyl benzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-24 to 2022-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-24 to 2022-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: “Regulation on Test Methods for Chemical Substances” Notification No. 2020 46 , National Institute of Environmental Research , Republic of Korea

Version / remarks:

2020-11-03

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Details on species / strain selection:

Sprague-Dawley rats are extensively used in toxicity studies for such as reproductive and developmental toxicity, and are selected because of the abundance of historical control data to compare.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks (P males), 12 weeks (P females)
- Weight at study initiation: 332.7 to 389.3 g (P males), 232.3 to 302.9 g (P females)
- Fasting period before study: No
- Housing: One animal/cage (during quarantine acclimation period and the dosing period), 1-3 animals/cage (during mating period) and one female + pups/cage (during lactation) in stainless wire mesh cages or in polycarbonate cages (from GD16 to necropsy day).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were acclimatized but the period was not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.3
- Humidity (%): 52.6-67.7
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 50 (main study males), from day 1 to approx. day 67 (main study females), from PND 1 to PND 4 or 13 (F1 generation), From day 1 to day 64 (recovery group males and females)

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test substance was weighed and placed in a container. The required amount of vehicle (required amount: 10 mL of vehicle for 1 kg of powder feed containing test substance), corn oil, was added and mixed using a vortex mixer until dissolved. The required amount of powder feed except for the amount of the test substance was weighed. The required amount of the test substance formulation and a small amount of powder feed were mixed in a container. Then, the mixture was placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5-10 minutes to yield the desired concentration. The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5-10 minutes for the control group.

DIET PREPARATION
- Rate of preparation of diet: Approx. weekly
- Mixing appropriate amounts with: Powder feed rodent chow (LabDiet ® CERTIFIED RODENT DIET 5002, powder type)
- Storage temperature of food: Under refrigeration conditions for 8 days, at room temperature (1 30°C) for 3 days.

VEHICLE
- Justification for use and choice of vehicle: Solubility properties
- Batch No: MKCN9742

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability analyses were conducted in a separate study (Study No B21450). The homogeneity and stability at room temperature (1-30°C) for 3 days and under refrigeration for 8 days for the dosing formulations comprising the dose levels of 0, 0.5 and 1.6% were confirmed.

Analyses of the dosing formulations were conducted in a separate study (Study No B21450). Samples were taken three times from the middle layer of each dosing formulation and analyzed for verification of dose concentration prior to dosing. As a result, the accuracies at 0.4 , 0.8 and 1.6% were 87.90, 95.76 and 93.63%. These results were within the acceptable range (range: ± 20% of nominal values).

Duration of treatment / exposure:

49 days (main study males and recovery group males and females), approx. 66 days (2 weeks prior to mating until post-partum day 13, main study females)

Frequency of treatment:

Continuously (feed)

Dose / conc.:

1 111.7 mg/kg bw/day (actual dose received)

Remarks:

Recovery group females (corresponding to 1.6% in feed)

Dose / conc.:

923.2 mg/kg bw/day (actual dose received)

Remarks:

Recovery group males (corresponding to 1.6% in feed)

Dose / conc.:

953.6 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 1.6% in feed

Dose / conc.:

469.6 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 0.8% in feed

Dose / conc.:

245 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 0.4% in feed

Dose / conc.:

1 294.5 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 1.6% in feed

Dose / conc.:

615.6 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 0.8% in feed

Dose / conc.:

338.7 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 0.4% in feed

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle (corn oil) in feed

No. of animals per sex per dose:

12 animals/sex/group (main study), 6 animals/sex/group for the control and high dose group (recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As a result of the 2-week dose range finding study (see IUCLID section 7.5.1), no test substance related effect was observed at 1.6%. Therefore, based on the dose range finding study, 1.6% was selected as the high dose of this study. The mid and low doses were selected at 0.8 and 0.4%, respectively, by applying a geometric ratio of 2.
- Rationale for animal assignment: Selected animals were randomly assigned to achieve even distribution of mean body weight of each group.
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight (approx. 18 hours)

Positive control:

No positive control was included in the study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily for clinical signs and twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the start of administration and once weekly thereafter
- Detailed clinical observations comprised: Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.), gait, posture and response to handling, and the presence of clonic or tonic movements, stereotypies (excessive grooming, repetitive circling, etc.) or bizarre behavior (self mutilation, walking backwar d, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of start of dosing (Day 1, prior to dosing) once a week during the dosing and recovery periods, and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

On the last day of the quarantine acclimation period vaginal smears were observed daily for females in order to examine estrous cycle for approximately 2 weeks before group assignment.

The estrus cycle for the females of the main groups were conducted from dosing initiation day to confirmed copulation day and necropsy day. Smears of vaginal mucosa were prepared in the morning daily. Prepared smears of the vaginal mucosa were stained with Diff Quick stain. Stained vaginal mucosa smears were examined using light microscopy. The estrus cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrus cycle was defined as the period between the day of estrus and prior to the day of next estrus.

Estrous cycle was calculated from dosing initiation day to day before mating initiation.

Sperm parameters (parental animals):

No

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- A maximum of 4 pups/litter/sex/litter (as nearly as possible) were randomly selected; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. External genitalia were examined grossly.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [sacrificed on Day 50]. All animals of the recovery groups were sacrificed 2 weeks after the final dosing.
- Maternal animals: All surviving animals [on Postpartum Day 14]. All animals of the recovery groups were sacrificed 2 weeks after the final dosing. Non copulated females were sacrificed on Day 26 after the last day of mating. Non delivered females were sacrificed on Gestation Day 26.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 5+6 were prepared for microscopic examination or weighed

Postmortem examinations (offspring):

SACRIFICE
- 4 male/female F1 offspring (if applicable) were sacrificed on postnatal day 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 5 were prepared for weighting. For all male and female neonates sacrificed on PND 13 were preserved only the thyroid and parathyroid glands in 10% neutral buffered formalin.

Statistics:

Statistical analysis was performed on the data of body weight, food consumption, estrous cycle, mating period, gestation period, post implantation loss rate, live birth index , mean litter size , body weights of pups, viability index on postnatal days 0 and 4, external examination, sex ratio, AGD index, nipple, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry , thyroid hormone and organ weights using SAS program. For the main groups and dosing period, the data was analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One way analysis of variance (ANOVA) was employed on homogeneous data (significance level: 0.05); then, if significant, Dunnett’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two tailed). Kruskal Wallis test was employed on heterogeneous data (significance level: 0.05); then, if significant, DSCF (Dwass Steel Critchlow Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two tailed). The data of mating index, fertility index and other data associated with gestation was analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the recovery groups, the data was analyzed utilizing Folded F test for homogeneity of variance (significance level: 0.05). Student’s t test was employed on homogeneous data or Aspin Welch t test was employed on heterogeneous data for confirming significance (significance levels: 0.05 and 0.01, two tailed).

Reproductive indices:

- Mating index (%) = (No. of females with evidence of mating/No. of females paired) × 100
- Mating period = Day of confirmed mating Day of initial mating ( based on the dosing day)
- Gestation period = Postpartum Day 0 Gestation Day 0 ( based on the dosing day)
- Male fertility index (%) = (No. of males impregnating a female / No. of males with confirmed mating) ×100
- Female fertility index (%) = (No. of pregnant females / No. of females with confirmed mating ) × 100
- Gestation index (%) = (No. of females with live pups / No. of pregnant females) × 100
- Post implantation loss rate (%) = No. of implantation No. of live pups) / No. of im plantation) × 100

Offspring viability indices:

- Mean litter size
- Live birth index (%) = (No of live pups on PND 0 / No. of implantation) × 100
- Viability index on postnatal d ay 0 (%) = (No. of pups born alive on PND 0 / Total No. of pups born) × 100
- Viability index on postnatal day 4 (%) = (No. of pups surviving on PND 4 / No. of pups born alive on PND 0) × 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Delayed delivery was observed in one female in the control group on gestation day 25 and dystocia with delayed delivery were observed in one female in the 0.4 and 0.8% groups on gestation day 25 (postpartum day 0), respectively.

This was not considered to be a test substance-related effect since this effect was observed in respectively only one female and thus considered to be incidental, observed in the control group, and showed no dose-dependence.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Sporadical changes with statistical significance were observed in body weight gain. These changes were temporary changes not related to feed intake, and it was considered not to be test substance-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Sporadical changes with statistical significance were not considered to be test substance-related since they were minior and not accompanied with body weight changes with statistical significance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All differences in clinical chemistry parameters were of small magnitude and even if statistically significant they were considered to represent normal biological variations. In addition, the variations were neither observed in both sexes nor confirmed in related parameters. Furthermore, some changes were observed in the recovery group only.

Endocrine findings:

no effects observed

Description (incidence and severity):

Sporadical statistical differences in thyroid hormone were considered to be of no toxicological significance because they were not dose-dependent, of small magnitude or not accompanied by any morphological changes of the organ.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal acute myometrial inflammation and severe dilatation in the uterus of one non-copulated female of the 0.8% dose group was observed, and the microscopic lesions corresponded to the macroscopic lesion in the uterus.

In the ovary, follicular cyst was observed in the ovary of each one of the two non-copulated female of the control and 1.6% dose groups, in the main group.

The lesions in the reproductive systems are not associated with the test substance, as they did not show significant differences in incidence between the control and 1.6% dose groups.

All other microscopic findings seen in various tissues and/or organs of all animals did not show significant differences in incidence compared to controls or were normal background lesions observed at the facility or were found with low/isolated frequency.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 294.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 953.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadical statistical differences in body weight of pups were considered to be of no toxicological relevance due to the missing correlation with the dose or unaccompanied with any related changes.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Sporadical statistical differences in thyroid hormone were considered to be of no toxicological significance because they were not dose-dependent, of small magnitude or not accompanied by any morphological changes of the organ.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1.6 other: %

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Corresponding to parental doses of 953.6 mg/kg bw/day (males) and 1294.5 mg/kg bw/day (females)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In a Combined Repeated Oral (Dietary) Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422, the no observed adverse effect level (NOAEL) of the test substance was 1.6% (corresponding to 953.6 mg/kg bw/day for males and 1294.5 mg/kg bw/day for females) in general toxicity, reproductive toxicity and developmental toxicity respectively, under the conditions of this study.

Executive summary:

The purpose of this study according to OECD guideline 422 and GLP was to determine the No Observed Adverse Effect Level (NOAEL) of the test substance for both general systemic and reproductive/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development as well as the neurotoxic potential of the test substance following repeated oral administration. In addition, some endocrine disruptor relevant endpoints were evaluated. A total of 4 groups were designated as follows:

Three groups were designated as Groups 2, 3 and 4 were treated with the test substance at dose levels of 0.4, 0.8 and 1.6% (the main group 245.0, 469.6 and 953.6 mg/kg bw/day for males and 338.7, 615.6 and 1294.5 mg/kg bw/day for females, respectively, also recovery group at 1.6% were 923.2 mg/kg bw/day for males, and 1111.7 mg/kg bw/day for females, respectively.) along with a control group, Group 1 (receiving the standard basal powder feed with corn oil). Each group consisted of 12 males and 12 females for the main study. Six animals were added to each group for the recovery groups, control (G1) and high dose (G4) groups. Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, observation of estrous cycle, reproductive function, neonate examination, functional observations, urinalysis, hematology, clinical chemistry, thyroid hormone analysis, gross post mortem examination, organ weights and histopathological evaluations of selected tissues. During the observation period, no deaths of animals or test substance-related clinical signs were observed in the 0.4, 0.8 and 1.6% groups for both sexes. No test substance-related effects were noted in the detailed clinical sign observations, body weight, food consumption, observation of estrous cycle, reproductive function, neonate examination, functional observations, urinalysis, hematology, clinical chemistry, thyroid hormone analysis, gross post mortem examination, organ weights and histopathological findings in the 0.4, 0.8 and 1.6% groups of both sexes.

 

Based on the results, the no observed adverse effect level (NOAEL) of the test substance was 1.6% (corresponding to 953.6 mg/kg bw/day for males and 1294.5 mg/kg bw/day for females) in general toxicity, reproductive toxicity and developmental toxicity respectively, under the conditions of this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016-07-29

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: “Regulation on Test Methods for Chemical Substances” Notification No. 2020 46 , National Institute of Environmental Research , Republic of Korea

Version / remarks:

2020-11-03

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl benzoate

EC Number:

202-284-3

EC Name:

Ethyl benzoate

Cas Number:

93-89-0

Molecular formula:

C9H10O2

IUPAC Name:

ethyl benzoate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Details on species / strain selection:

Sprague-Dawley rats are extensively used in toxicity studies for such as reproductive and developmental toxicity, and are selected because of the abundance of historical control data to compare.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks (P males), 12 weeks (P females)
- Weight at study initiation: 332.7 to 389.3 g (P males), 232.3 to 302.9 g (P females)
- Fasting period before study: No
- Housing: One animal/cage (during quarantine acclimation period and the dosing period), 1-3 animals/cage (during mating period) and one female + pups/cage (during lactation) in stainless wire mesh cages or in polycarbonate cages (from GD16 to necropsy day).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were acclimatized but the period was not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.3
- Humidity (%): 52.6-67.7
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 50 (main study males), from day 1 to approx. day 67 (main study females), from PND 1 to PND 4 or 13 (F1 generation), From day 1 to day 64 (recovery group males and females)

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was chosen as it is a possible route of exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test substance was weighed and placed in a container. The required amount of vehicle (required amount: 10 mL of vehicle for 1 kg of powder feed containing test substance), corn oil, was added and mixed using a vortex mixer until dissolved. The required amount of powder feed except for the amount of the test substance was weighed. The required amount of the test substance formulation and a small amount of powder feed were mixed in a container . Then, the mixture was placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5-10 minutes to yield the desired concentration. The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5-10 minutes for the control group.

DIET PREPARATION
- Rate of preparation of diet: Approx. weekly
- Mixing appropriate amounts with: Powder feed rodent chow (LabDiet ® CERTIFIED RODENT DIET 5002, powder type)
- Storage temperature of food: Under refrigeration conditions for 8 days, at room temperature (1 30°C) for 3 days.

VEHICLE
- Justification for use and choice of vehicle: Solubility properties
- Batch No: MKCN9742

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability analyses were conducted in a separate study (Study No B21450). The homogeneity and stability at room temperature (1-30°C) for 3 days and under refrigeration for 8 days for the dosing formulations comprising the dose levels of 0, 0.5 and 1.6% were confirmed.

Analyses of the dosing formulations were conducted in a separate study (Study No B21450). Samples were taken three times from the middle layer of each dosing formulation and analyzed for verification of dose concentration prior to dosing. As a result, the accuracies at 0.4, 0.8 and 1.6% were 87.90, 95.76 and 93.63%. These results were within the acceptable range (range: ± 20% of nominal values).

Duration of treatment / exposure:

49 days (main study males and recovery group males and females), approx. 66 days (2 weeks prior to mating until post-partum day 13, main study females)

Frequency of treatment:

Continuously (feed)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 111.7 mg/kg bw/day (actual dose received)

Remarks:

Recovery group females (corresponding to 1.6% in feed)

Doses / concentrations 2

Dose / conc.:

923.2 mg/kg bw/day (actual dose received)

Remarks:

Recovery group males (corresponding to 1.6% in feed)

Doses / concentrations 3

Dose / conc.:

953.6 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 1.6% in feed

Doses / concentrations 4

Dose / conc.:

469.6 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 0.8% in feed

Doses / concentrations 5

Dose / conc.:

245 mg/kg bw/day (actual dose received)

Remarks:

In males, corresponding to 0.4% in feed

Doses / concentrations 6

Dose / conc.:

1 294.5 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 1.6% in feed

Doses / concentrations 7

Dose / conc.:

615.6 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 0.8% in feed

Doses / concentrations 8

Dose / conc.:

338.7 mg/kg bw/day (actual dose received)

Remarks:

In females, corresponding to 0.4% in feed

Doses / concentrations 9

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle (corn oil) in feed

No. of animals per sex per dose:

12 animals/sex/group (main study), 6 animals/sex/group for the control and high dose group (recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As a result of the 2-week dose range finding study (see IUCLID section 7.5.1), no test substance related effect was observed at 1.6%. Therefore, based on the dose range finding study, 1.6% was selected as the high dose of this study. The mid and low doses were selected at 0.8 and 0.4%, respectively, by applying a geometric ratio of 2.
- Rationale for animal assignment: Selected animals were randomly assigned to achieve even distribution of mean body weight of each group.
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight (approx. 18 hours)

Positive control:

No positive control was included in the study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily for clinical signs and twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the start of administration and once weekly thereafter
- Detailed clinical observations comprised: Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.), gait, posture and response to handling, and the presence of clonic or tonic movements, stereotypies (excessive grooming, repetitive circling, etc.) or bizarre behavior (self mutilation, walking backwar d, etc.)

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of start of dosing (Day 1, prior to dosing) once a week during the dosing and recovery periods, and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning prior to necropsy
- Anaesthetic used for blood collection: Yes (isoflurane) 
- Animals fasted: Yes (except neonates, overnight for approx. 18 hours)
- How many animals: All surviving animals  
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In the morning prior to necropsy
- Animals fasted: Yes (except neonates, overnight for approx. 18 hours)
- How many animals: All surviving animals  
- Parameters checked in table 3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes 
- Time of blood sample collection: In the morning prior to necropsy
- Animals fasted: Yes (overnight for approx. 18 hours)
- How many animals: Pups on PND 13 and for all adult males 

URINALYSIS: Yes 
- Time schedule for collection of urine: Urinalysis was performed on 6 males in each group in the main group and all surviving males in the recovery group a few days before necropsy.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (Fresh urine (collected urine for about 3 hours) and collected urine for about 24 hours were examined. When fresh urine was collected, feeding and dosing were not performed. However, drinking water was provided ad libitum.)
- Parameters checked in table 1 were examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observations were conducted on six males and females per group in the main group and all surviving animals in the recovery group a few days before necropsy.
- Dose groups that were examined: Six males and females per group in the main group and all surviving animals in the recovery group
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Organs weighted are listed in table 5

HISTOPATHOLOGY: Yes, tissues in Table 6 were prepared for microscopic examination

Optional endpoint(s):

Optional endpoints: Yes, reproductive toxicity endpoints (see IUCLID section 7.8.1)

Other examinations:

Yes, reproductive toxicity examinations (see IUCLID section 7.8.1)

Statistics:

Statistical analysis was performed on the data of body weight, food consumption, estrous cycle, mating period, gestation period, post implantation loss rate, live birth index , mean litter size , body weights of pups, viability index on postnatal day s 0 and 4, external examination, sex ratio, AGD index, nipple functional observations (hindlimb landing foot splay, grip strength and motor activity) activity), urine volume, hematology, clinical chemistry , thyroid hormone and organ weights using SAS program. For the main groups and dosing period, the data was analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One way analysis of variance (ANOVA) w as employed on homogeneous data (significance level: 0.05); then, if significant, Dunnett’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two tailed). Kruskal Wallis test was employed on heterogeneous data (significance level: 0.05); then, if significant, DSCF (Dwass Steel Critchlow Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two tailed). The data of mating index, fertility index and other data associated with gestation was analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the recovery groups, the data was analyzed utilizing Folded F test for homogeneity of variance (significance level: 0.05). Student’s t test was employed on homogeneous data or Aspin Welch t test was employed on heterogeneous data for confirming significance (significance levels: 0.05 and 0.01, two tailed).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Delayed delivery was observed in one female in the control group on gestation day 25 and dystocia with delayed delivery were observed in one female in the 0.4 and 0.8% groups on gestation day 25 (postpartum day 0), respectively.

This was not considered to be a test substance-related effect since this effect was observed in only one female in the respective group, was also observed in the control group, and showed no dose-dependence. Thus, it was considered to be incidentally.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Sporadical changes with statistical significance were observed in body weight gain. These changes were temporary changes not related to feed intake, and it was considered not to be test substance-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Sporadical changes with statistical significance were not considered to be test substance-related changes since they were minior changes and not accompanied body weight changes with statistical significance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All differences in clinical chemistry parameters were of small magnitude and even if statistically significant they were considered to represent normal biological variations. In addition, the variations were neither observed in both sexes nor confirmed in related parameters. Furthermore, some changes were observed in the recovery group only.

Endocrine findings:

no effects observed

Description (incidence and severity):

Sporadical statistical differences in thyroid hormone were considered to be of no toxicological significance because they were not dose-dependent, of small magnitude or not accompanied by any morphological changes of the organ.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Sporadical statistical differences in organ weights were considered to be of no toxicological significance because they were not dose-dependent, of small magnitude or unaccompanied any morphological changes of the organ.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Reduction in size was observed in the testis of one male of the control group in the main group.
Dilatation of uterus and vagina was also noted in one female of the control group in the main group.
Yellowish and serous fluid in the uterus was observed in one non-copulated female of the 0.8% group in the main group.

Other macroscopic findings seen in various tissues and/or organs of all animals did not show significant differences in incidence compared to controls or were normal background lesions observed at this facility or were found with low/isolated frequency.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal acute myometrial inflammation and severe dilatation in the uterus of one non-copulated female of the 0.8% dose group was observed, and the microscopic lesions corresponded to the macroscopic lesion in the uterus.

In the ovary, follicular cyst was observed in the ovary of each one of the two non-copulated female of the control and 1.6% dose groups, in the main group.

The lesions in the reproductive systems are not associated with the test substance, as they did not show significant differences in incidence between the control and 1.6% dose groups.

All other microscopic findings seen in various tissues and/or organs of all animals did not show significant differences in incidence compared to controls or were normal background lesions observed at the facility or were found with low/isolated frequency.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 953.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 294.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a Combined Repeated Oral (Dietary) Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test according to OECD guideline 422, the no observed adverse effect level (NOAEL) of the test substance was 1.6% (corresponding to 953.6 mg/kg bw/day for males and 1294.5 mg/kg bw/day for females) in general toxicity, reproductive toxicity and developmental toxicity respectively, under the conditions of this study.

Executive summary:

The purpose of this study according to OECD guideline 422 and GLP was to determine the No Observed Adverse Effect Level (NOAEL) of the test substance for both general systemic and reproductive/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development as well as the neurotoxic potential of the test substance following repeated oral administration. In addition, some endocrine disruptor relevant endpoints were evaluated. A total of 4 groups were designated as follows:

Three groups were designated as Groups 2, 3 and 4 were treated with the test substance at dose levels of 0.4, 0.8 and 1.6% (the main group 245.0, 469.6 and 953.6 mg/kg bw/day for males and 338.7, 615.6 and 1294.5 mg/kg bw/day for females, respectively, also recovery group at 1.6% were 923.2 mg/kg bw/day for males, and 1111.7 mg/kg bw/day for females, respectively.) along with a control group, Group 1 (receiving the standard basal powder feed with corn oil). Each group consisted of 12 males and 12 females for the main study. Six animals were added to each group for the recovery groups, control (G1) and high dose (G4) groups. Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, observation of estrous cycle, reproductive function, neonate examination, functional observations, urinalysis, hematology, clinical chemistry, thyroid hormone analysis, gross post mortem examination, organ weights and histopathological evaluations of selected tissues. During the observation period, no deaths of animals or test substance-related clinical signs were observed in the 0.4, 0.8 and 1.6% groups for both sexes. No test substance-related effects were noted in the detailed clinical sign observations, body weight, food consumption, observation of estrous cycle, reproductive function, neonate examination, functional observations, urinalysis, hematology, clinical chemistry, thyroid hormone analysis, gross post mortem examination, organ weights and histopathological findings in the 0.4, 0.8 and 1.6% groups of both sexes.

 

Based on the results, the no observed adverse effect level (NOAEL) of the test substance was 1.6% (corresponding to 953.6 mg/kg bw/day for males and 1294.5 mg/kg bw/day for females) in general toxicity, reproductive toxicity and developmental toxicity respectively, under the conditions of this study.