Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

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:

25 March 2021 to 23 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

EC Number:

951-458-1

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

Test material form:

liquid

Details on test material:

Substance name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Test item handling: Use amber glassware or wrap container in aluminum foil
EC Number: 951-458-1
Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Specific details on test material used for the study:

Physical Description: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Substance Name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Chemical name (IUPAC): 2,2-bis[({3-[methyl(phenyl)amino]propanoyl}oxy)methyl]butyl 3-[methyl(phenyl)amino]propanoate
EC number: 951-458-1
Molecular formula: EtC(CH2OCOCH2CH2NMePh)3
Molecular weight: 617.79 g/mol
Specific gravity / density: 1.08 at 25°C

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: males were 10-11 weeks old, females were 13-14 weeks old
- Weight at study initiation: males weighed between 282 and 325 g, females weighed between 196 and 247 g.
- Fasting period before study: not reported
- Housing: polycarbonate cages (Macrolon, MIV type, height 18 cm).
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY: It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21 °C
- Humidity (%): 47 to 73%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 25 March 2020 To: 29 September 2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Due to the viscosity of the test item, formulations were blended using the T25 Ultra Turrax homogenizer with the S25N-18G unit at a minimum of 9000 rpm for 10 minutes, followed by 30 minutes stirring at room temperature.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): not reported
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select a suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 100, 300 and 1000 mg/kg/day
- Amount of vehicle (if gavage): not reported
- Lot/batch no. (if required): not reported
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

Duration of treatment / exposure:

Minimum 28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000

Remarks:

mg/kg

Doses / concentrations 2

Dose / conc.:

300

Remarks:

mg/kg

Doses / concentrations 3

Dose / conc.:

100

Remarks:

mg/kg

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of the test item in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: F0-males (except for animals which were sacrificed in extremis) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- Rationale for selecting satellite groups: not reported
- Post-exposure recovery period in satellite groups: not reported
- Section schedule rationale (if not random):
- Other:

Positive control:

Not reported

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: once daily
- Cage side observations checked in table [No.?] were included: not reported

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- 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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: not specified
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: F0-males (except for animals which were sacrificed in extremis) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 80
- Parameters checked in table [No.?] were examined: not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: not specified
- Animals fasted: F0-males (except for animals which were sacrificed in extremis) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 80
- Parameters checked in table [No.?] were examined: not specified

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: not specified
- Animals fasted: F0-males (except for animals which were sacrificed in extremis) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 80

URINALYSIS: Yes
- Time schedule for collection of urine: not specified
- Metabolism cages used for collection of urine: Yes
- Animals fasted: not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

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% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.

Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex) and clotting parameters).

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related changes in body weights and body weight gain were observed at 1000 mg/kg/day.
Mean body weight was decreased (down to 0.91x of control) in males at 1000 mg/kg/day during the complete treatment period; mean body weight gain was decreased (down to 0.60x of control) at the end of treatment.
Mean body weight (not statistically significant) and body weight gain were decreased (0.93x and 0.84x of control, respectively) in females at 1000 mg/kg/day on post coitum Day 20.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes in food consumption before or after correction for body weight were observed at 1000 mg/kg/day in males and females.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters of treated rats were affected by treatment with the test item at 100, 300 and 1000 mg/kg/day.

The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise.

• Mean red blood cell count (RBC) was decreased: 0.81x, 0.78x and 0.62x of control in males at 100, 300 and 1000 mg/kg/day, respectively, and 0.89x and 0.76x of control in females at 300 and 1000 mg/kg/day, respectively.

• Mean reticulocyte concentration (RETIC) was increased: 1.90x, 2.84x and 4.14x of control in males at 100, 300 and 1000 mg/kg/day, respectively (not always statistically significant), and 1.70x and 3.18x of control in females at 300 and 1000 mg/kg/day, respectively (not always statistically significant).

• Mean percentage red blood cell distribution width gated (RDWG) was increased: 1.17x and 1.25x of control in males at 300 and 1000 mg/kg/day, respectively.

• Mean hemoglobin concentration (HGB) was decreased: 0.84x, 0.84x and 0.79x of control in males at 100, 300 and 1000 mg/kg/day, respectively, and 0.91x of control in females at 1000 mg/kg/day.

• Mean hematocrit concentration (HCT) was decreased: 0.87x, 0.91x and 0.84x of control in males at 100, 300 and 1000 mg/kg/day, respectively.

• Mean of mean corpuscular volume (MCV) was increased: 1.08x, 1.17x and 1.35x of control in males at 100, 300 and 1000 mg/kg/day, respectively, and 1.11x and 1.30x of control in females at 300 and 1000 mg/kg/day, respectively.

• Mean of mean corpuscular hemoglobin (MCH) was increased: 1.09x and 1.27x of control in males at 300 and 1000 mg/kg/day, respectively, and 1.19x of control in females at 1000 mg/kg/day.

• Mean of mean corpuscular hemoglobin concentration (MCHC) was decreased: 0.97x, 0.93x and 0.93x of control in males at 100, 300 and 1000 mg/kg/day, respectively, and 0.97x and 0.91x of control in females at 300 and 1000 mg/kg/day, respectively.

• Mean platelet (PLT) concentration was decreased: 0.69x of control in females at 1000 mg/kg/day.

Any other statistically significant changes in hematological parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were affected by treatment with the test item at 100, 300 and 1000 mg/kg/day.

The following changes distinguished treated from control animals. The differences were statistically significant unless indicated otherwise.

• Mean total protein (TPROT) concentration was decreased: 0.93x, 0.90x and 0.90x of control in males at 100, 300 and 1000 mg/kg/day, respectively.

• Mean albumin (ALB) concentration was decreased: 0.94x, 0.93x and 0.95x of control in males at 100, 300 and 1000 mg/kg/day, respectively.

• Mean total bilirubin (TBIL) concentration was increased: 3.23x of control in males and 7.87x of control in females at 1000 mg/kg/day (not statistically significant in females).

• Mean alanine aminotransferase (ALT) concentration was decreased: 0.51x of control in females at 1000 mg/kg/day.

• Mean urea concentration was decreased: 0.65x of control in females at 1000 mg/kg/day.

• Mean glucose (GLUC) concentration was increased: 1.68x of control in females at 1000 mg/kg/day.

• Mean cholesterol (CHOL) concentration was decreased: 0.61x of control in females at 1000 mg/kg/day.

• Mean calcium (CA) concentration was increased: 1.05x of control in females at 1000 mg/kg/day.

• Mean inorganic phosphate (PHOS) concentration was increased: 2.20x of control in females at 1000 mg/kg/day.

Any other statistically significant changes in clinical chemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend or an opposite (i.e. increase) would be expected in case of target organ toxicity.

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Some organ weight differences (males: absolute adrenal glands and relative liver and kidney weights; females: relative brain weight) were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Spleen: There was a dose-related increase in incidence and severity of extramedullary hematopoiesis (up to marked degree in males and up to massive degree in females) and congestion (up to moderate degree) in the spleen of males and females, starting at 100 mg/kg/day. In females, an additional dose-related increase in severity of pigmentation (black-brown) in macrophages was observed.

Liver: Extramedullary hematopoiesis was observed at minimal degree in males starting at 100 mg/kg/day and in females up to slight degree, starting at 300 mg/kg/day. Pigmentation in Kupffer cells/macrophages (brown) was observed in males up to slight degree and in females at minimal degree, starting at 300 mg/kg/day.

Bone marrow: There was a dose-related increase in incidence and severity of increased cellularity (mainly hematopoietic cells) up to slight degree in males (starting at 100 mg/kg/day) and females (starting at 300 mg/kg/day).

Kidney: Accumulation of pigment (yellow-brown) up to slight degree, was observed in the cortical tubular epithelium of females treated at 300 and 1000 mg/kg/day.

Adrenal glands: Extramedullary hematopoiesis was observed in two females treated at 1000 mg/kg/day.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw (total dose)

System:

hepatobiliary

Organ:

adrenal glands

bone marrow

kidney

liver

spleen

Treatment related:

yes

Any other information on results incl. tables

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day (for deviation see Appendix 8). The rats of the control group received the vehicle, polyethylene glycol 400, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-64 days). Females that failed to deliver offspring or had a total litter loss were treated for 42-54 days.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results
Parental toxicity was observed starting at 100 mg/kg/day in males and at 300 mg/kg/day in females.

Yellow discolored urine was observed during the daily detailed clinical observations in one female at 300 mg/kg/day on a single day and all females at 1000 mg/kg/day from Week 6 onwards. In addition, yellow-brown tubular pigment was seen in the kidneys of females at 300 and 1000 mg/kg/day, which was without additional degenerative findings. Since the used test item was a light orange liquid and females of these dose groups showed yellow discolored urine, the observed discolored urine and tubular pigment was most likely related to the (excretion of) colored test item. These findings were therefore considered as non-adverse.

Body weight was decreased in males at 1000 mg/kg/day from premating Day 8 until the end of treatment. In addition, food consumption was decreased in males at 1000 mg/kg/day during the premating period. At the magnitude of these effects, these changes were considered as adverse.

Body weight was decreased in females at 1000 mg/kg/day on post-coitum Day 20. In addition, food consumption was decreased in females at 1000 mg/kg/day between Lactation Days 1-7. These changes were most likely caused by the lower pup weights at 1000 mg/kg/day rather than by direct systemic toxicological effects resulting from treatment with the test item.

A significant increase in (extramedullary) hematopoiesis was observed in mainly spleen (together with congestion) and bone marrow (starting at 100 mg/kg/day in males and females), and to a lesser extend in liver (starting at 100 mg/kg/day in males and at 300 mg/kg/day in females) and adrenal glands (at 1000 mg/kg/day in females). This resulted in enlargement and black-brown discoloration of the spleen and increased weights of the spleen. Furthermore, pigment was observed in Kupffer cells/macrophages of the liver and in macrophages of the spleen (both sexes). This pigment is considered to represent hemosiderin, which is a form of iron storage from destructed red blood cells.

The increased hematopoiesis and deposition of black-brown pigmented macrophages reflected changes in hematology and clinical biochemistry values, consisting of a significant decrease in red blood cell count (starting at 100 mg/kg/day in males and at 300 mg/kg/day in females), hematocrit (starting at 100 mg/kg/day in males) and hemoglobin concentrations (starting at100 mg/kg/day in males and at 1000 mg/kg/day in females) and a massive increase in reticulocytes (starting at 100 mg/kg/day in males and 300 mg/kg/day in females) and total bilirubin concentrations (at 1000 mg/kg/day in males and females). The increase in percentage red blood cell distribution width (RDWG; starting at 300 mg/kg/day in males), mean corpuscular volume (MCV; starting at 100 mg/kg/day in males and at 300 mg/kg/day in females), mean corpuscular hemoglobin (MCH) content (starting at 300 mg/kg/day in males and at 1000 mg/kg/day in females) and decreased mean corpuscular hemoglobin concentration (MCHC; starting at 100 mg/kg/day in males and at 300 mg/kg/day in females) may be attributed to the increase of a heterogeneous reticulocyte population in the blood.

The cause of the decreased red blood cells was not clear. There were no indications for hemorrhages or inflammatory responses, suggesting that the test item may affect the red blood cells directly. Normally, small decreases in hematological parameters can be compensated by a mild increase in hematopoiesis without effecting the well-being, growth, development or life span of an animal. However, in the present study, the effects especially on spleen and hematology parameters were quite drastic (in males at all doses and in females to a lesser extend starting at 300 mg/kg/day), suggesting that in these dose groups the animals are not capable of compensating the test item-induced red blood cell loss. Therefore, the findings in spleen and hematology parameters were considered adverse in male rats at all doses and in females starting at 300 mg/kg/day.

The decreased plasma total protein and albumin concentrations in males at 100, 300 and 1000 mg/kg/day were considered as non-adverse since these changes were not associated with any adverse pathological alterations.

A marked test item-related decrease in serum total T4 levels was noted in males (starting at 300 mg/kg/day) and females (at 1000 mg/kg/day).

None of the pregnant females treated at 1000 mg/kg/day had healthy offspring, which resulted in total litter losses between 2 and 9 days after delivery. The poor health of the pups could not be explained by any morphological abnormalities in the reproductive organs but may well be indirectly related to the significant changes in the red blood cell population.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex) and clotting parameters).

Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No treatment-related toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results
No developmental toxicity was observed up to 300 mg/kg/day.

The post-implantation survival and live birth indices, and litter size were decreased at 1000 mg/kg/day. In addition, one or more severe clinical signs, such as a pale, lean and/or dehydrated appearance, hunched posture, dry skin and/or low body temperature (cold) were noted in pups at 1000 mg/kg/day. As a consequence, all pups at 1000 mg/kg/day went missing, were found dead or were euthanized in extremis between PND 1 to 9, resulting in decreased viability and lactation indices at 1000 mg/kg/day. Moreover, body weights of pups at 1000 mg/kg/day between PND 1 and 7 were decreased, indicative of a growth retardation, which as a result may have also lead to smaller anogenital distances of male pups at 1000 mg/kg/day.

Taken together, these findings were considered as adverse.

No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, duration of gestation, parturition, sex ratio, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The developmental toxicity observed in this study occurred at the dose level associated with severe parental toxicity at 1000 mg/kg/day (i.e. loss of red blood cells and a massive increase in reticulocytes). It could not be excluded that these effects resulted in the observed developmental toxicity.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for the test item were established:

Parental NOAEL: no NOAEL could be established (based on the macroscopic and microscopic findings in the spleen as well as a subset of hematology and clinical biochemistry parameters starting at 100 mg/kg/day in males and at 300 mg/kg/day in females).

Reproduction NOAEL: at least 1000 mg/kg/day.

Developmental NOAEL: 300 mg/kg/day (based on the severe clinical signs, decreased litter size, pup body weights, anogenital distance in male pups, post-implantation survival, live birth, viability and lactation indices at 1000 mg/kg/day).

Executive summary:

The objectives of this study were to determine the potential toxic effects of the test item when given orally by gavage for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the Dose Range Finder.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups).

The developmental toxicity observed in this study occurred at the dose level associated with severe parental toxicity at 1000 mg/kg/day (i.e. loss of red blood cells and a massive increase in reticulocytes). It could not be excluded that these effects resulted in the observed developmental toxicity.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) for the test item were established:

Parental NOAEL: no NOAEL could be established (based on the macroscopic and microscopic findings in the spleen as well as a subset of hematology and clinical biochemistry parameters starting at 100 mg/kg/day in males and at 300 mg/kg/day in females).

Reproduction NOAEL: at least 1000 mg/kg/day.

Developmental NOAEL: 300 mg/kg/day (based on the severe clinical signs, decreased litter size, pup body weights, anogenital distance in male pups, post-implantation survival, live birth, viability and lactation indices at 1000 mg/kg/day).