Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

EC Number:

614-257-7

Cas Number:

68071-40-9

Molecular formula:

C18H34O4, C20H38O4 and C22H42O4 (mainly)

IUPAC Name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Specific details on test material used for the study:

Identification: Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs
Batch (Lot) Number: 190708588
Expiry date: 31 July 2020 (expiry date)
Physical Description: Transparent liquid
Purity/Composition: See Certificate of Analysis , UVCB
Storage Conditions: At room temperature protected from light

Test Facility Test Item Number: 210500/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or trade name): 2-Propenoic acid, ester with C12-16-alkyl glycidyl ether
CAS number: 68071-40-9
EC number: 614-257-7
Molecular weight: ≥ 314.46 - ≤ 370.57
Specific gravity / density: Not available
Solubility in vehicle: Water: ~1.9 mg/L (according to OECD TG 105, REACH registration dossier)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. 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. Charles River Den Bosch has general and reproduction/developmental historical 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it
does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
On 18 Dec 2019, female Crl: WI(Han) rats were received and on 30 Dec 2019, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 10-11 weeks old and weighed between 301 and 355 g and females were 13-14 weeks old and weighed between 198 and 253 g. A health inspection was performed before the initiation of dosing.

A total of 40 females was selected at randomization before initiation of the pretest phase. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males).

On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment,bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.

Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20-22°C with an actual daily mean relative humidity of 32 to 53%. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a 14-day dose range finder with oral administration of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs in rats (Test Facility Reference No. 20212887), and in an attempt to produce graded responses to the test item.

Vehicle:

corn oil

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. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. No adjustment was made for specific gravity of the test item. Adjustment was made for specific gravity of the vehicle. No aAdjustment was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn oil. Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment was complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 20212886.

- Concentration in vehicle: 0, 20, 60, 200 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis. All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20149504).

Duplicate sets of samples (approximately 500 mg accurately weighed) 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.

Duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20149504) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20149504.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Duration of treatment / exposure:

Males were treated for 28-29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-65 days (most females), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy.

Frequency of treatment:

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle (corn oil)

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 male/10 female/per dose (0, 100, 300, 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Detection of mating was not confirmed in first instance for female no. 45. Evidence of mating was obtained by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the
mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed directly after dosing based on the results of the dose range finder

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

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, Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Yes, see attached study report)
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due
to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0 females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single
pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of
the necropsy procedure.

CLINICAL CHEMISTRY: Yes (see attached study report)
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due
to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0 females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single
pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of
the necropsy procedure.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
F0-generation: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 12-14) These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).The following tests were performed:
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 =
abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per
animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light
conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway,
USA). Total movements and ambulations were reported. Ambulations represent
movements characterized by a relocation of the entire body position like walking,
whereas total movements represent all movements made by the animals, including
ambulations but also smaller or more fine movements like grooming, weaving or
movements of the head.

IMMUNOLOGY: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table in attached study report)

HISTOPATHOLOGY: Yes (see table i attached study report)

Other examinations:

Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

All male pups in each litter were examined for the number of areola/nipples on PND 13.

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 comparison 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 findings were noted during the weekly arena observations in this study.

Salivation was observed in males starting at 100 mg/kg/day, and in females starting at 300 mg/kg/day. Salivation was most extensive at 1000 mg/kg/day, observed from Day 6 of treatment onwards (both sexes). This sign was considered to be a physiological response than a sign of systemic toxicity.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In males, no mortality occurred during the study period.

Two females at 1000 mg/kg/day (Nos. 72 and 74) were sacrificed in extremis on post coitum Day 23 with delivery difficulties.
Female No. 72 had an incomplete delivery. Eight pups were born, of which one was alive, and two dead pups were present in the uterus (all without abnormalities). Microscopic findings that were regarded secondary to the presence of dead fetuses in the uterus were recorded in: uterus (moderate mixed cell inflammation); liver (moderate multifocal coagulative necrosis, correlating to the necropsy finding ‘pale discoloration’); spleen (moderate extramedullary hematopoiesis) and bone marrow (slight increased hematopoietic cellularity) which was most likely related to blood loss; and in thymus and mesenteric lymph node (slight lymphoid depletion) most likely related to poor condition/stress.
In female No. 74 ten dead and five living pups were present in the uterus (all live pups without abnormalities). Piloerection was observed on the day of sacrifice. At microscopic examination findings were recorded in: liver (moderate multifocal coagulative necrosis), most likely related to the presence of dead fetuses in the uterus; spleen (moderate extramedullary hematopoiesis), regarded most likely related to blood loss; and in thymus and lymph node (moderate lymphoid depletion/lymphocytolysis and slight lymphoid depletion respectively), most likely related to poor condition/stress.

These premature decedents are unlikely to be test item-related, but considered to be related to the delivery difficulties resulting in the presence of dead fetuses in the uterus with secondary coagulative necrosis in liver.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item up to 1000 mg/kg/day.
Any statistically significant changes in body weight gain in females during the post coitum period were considered to be unrelated to treatment since no dose-related trend was apparent and mean values remained within the historical control range.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in haematology parameters.

Any changes in haematology parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of change and/or absence of a dose response, and with mean values remaining within normal ranges of biological variation.

Coagulation parameters of treated rats were considered not to have been affected by treatment. Increased APTT levels in males at 100 and 300 mg/kg/day were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend, and with mean values remaining within the historical control range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Coagulation parameters of treated rats were considered not to have been affected by treatment. Increased APTT levels in males at 100 and 300 mg/kg/day were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend, and with mean values remaining within the historical control range .

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Any (statistically significant) changes in clinical chemistry parameters were considered to be unrelated to administration of the test item due to the minimal magnitude of change and/or absence of a dose response, and with mean values remaining within normal ranges of biological variation.

Thyroid hormone analyses:
In males at 1000 mg/kg/day total T4 and TSH levels were increased with statistical significance (1.26x and 1.60x of control, respectively), which was considered treatment related. Mean values were on the upper limit ofor above the historical control range. In females no toxicologically relevant changes in total T4 or TSH levels were recorded.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment up to 1000 mg/kg/day.

In treated males at all dose levels, grip strength of the foreleg was decreased with statistical significance compared with concurrent controls. As control values were slightly high, mean values remained within the historical control range, no dose-relation was apparent and no clinical signs were observed suggesting decreased muscle strength, these changes were considered not test item-related.
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. In males at 1000 mg/kg/day, total movements and ambulations were slightly increased compared with concurrent controls, but with high variation at the individual level. As all mean values remained within the historical control range , no toxicological relevance was attributed to this finding.

In females, motor activity was similar between treated and control females. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant, test item related higher liver weights were recorded in both sexes at 1000 mg/kg/day.

The statistically significant higher liver weights at 1000 mg/kg/day (in males only statistically significant relative to body weights) correlated to the microscopic finding centrilobular hepatocellular hypertrophy in males, and the increased incidence and severity of cytoplasmic rarefaction (most likely glycogen) in females.

There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related macroscopic findings were present in the liver of males and consisted of an increased incidence of accentuated lobular pattern at 1000 mg/kg/day. This was recorded in 2/10 males for each the control, 100 and 300 mg/kg/day groups, and in 6/10 males at 1000 mg/kg/day. There were no test item related macroscopic findings in female rats; the single female at 1000 mg/kg/day with accented lobular pattern of the liver was considered to be within background.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Test item related microscopic findings at the end of the treatment period were noted in the liver of both sexes and thyroid glands of males.

Liver: In two males at 300 mg/kg/day and eight males at 1000 mg/kg/day centrilobular hepatocellular hypertrophy was recorded at minimal degree. This finding correlated to the accentuated lobular pattern recorded at necropsy and the increased liver weights at 1000 mg/kg/day.
In females at 1000 mg/kg/day an increased incidence and severity of cytoplasmic rarefaction (most likely glycogen) was recorded at minimal or slight degree. This was regarded the microscopic correlate to the increased liver weights in this dose group.

Thyroid glands: In males at 1000 mg/kg/day a minor increase in incidence and severity of follicular cell hypertrophy was recorded at minimal or slight degree. A background level of minimal follicular cell hypertrophy was recorded in the remaining dose groups including controls.

The remainder of the recorded microscopic findings at scheduled sacrifices 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The following was concluded for systemic toxicity:

Parental (systemic) NOAEL: 1000 mg/kg/day (Note: one female at 1000 mg/kg/day was found dead on Day 16 of treatment, however as a relation to test item could not be confirmed this mortality was not taken into account when determining the parental NOAEL).

Applicant's summary and conclusion

Conclusions:

Wistar Han rats were treated with Reaction product of Reaction Product of 2-Propenoic Acid and Oxirane, Mono[(C12-16-Alkyloxy)Methyl] Derivs (PE120) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day in accordance to OECD 422. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for reproduction was evaluated to be1000 mg/kg bw/day. For developmental toxicity, NOAEL wa established at 300 mg/kg bw/day based on delivery difficulties in 2/10 high dose females resulting in decreased gestation index and post implantation survival index.

Executive summary:

The objectives of this OECD 422 study were to determine the potential repeated dose toxicity as well as reproductive and developmental toxicity of Reaction Product of 2-Propenoic Acid and Oxirane, Mono[(C12-16-Alkyloxy)Methyl] Derivs(PE120) when given orally by gavage for a minimum of 28 days to Wistar Han rats, evaluating male and female reproductive performance such as gonadal function, mating behaviour, 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 0, 100, 300 and 1000 mg/kg/day, based on the results of a preliminary dose range finding study. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

Two females at 1000 mg/kg/day were sacrificedin extremis on post‑coitum Day 23 with delivery difficulties and dead pups in the uterus. Microscopic findings that were regarded secondary to the presence of dead fetuses in the uterus were recorded in: uterus (moderate mixed cell inflammation); liver (moderate multifocal coagulative necrosis, correlating to pale discoloration in one female); spleen (moderate extramedullary hematopoiesis) and/or bone marrow (slight increased hematopoietic cellularity) which was most likely related to blood loss; and in thymus (slight/moderate lymphoid depletion and/or lymphocytolysis) and mesenteric lymph node (slight lymphoid depletion) most likely related to poor condition/stress. These premature decedents are unlikely to be test item‑related, but considered to be a consequence of the delivery dose‑related difficulties resulting in the presence of dead fetuses in the uterus with secondary coagulative necrosis in liver.

Non-adverse findings were present in the liver of both sexes (males starting at 300 mg/kg/day, and females at 1000 mg/kg/day). In males starting at 300 mg/kg/day, centrilobular hepatocellular hypertrophy, increased liver weights and accentuated lobular pattern were observed. In females at 1000 mg/kg/day increased incidence/severity of cytoplasmic rarefaction and increased liver weights were recorded. In males at 1000 mg/kg/day,minor increased incidence and severity of follicular cell hypertrophy in the thyroid gland wererecorded.

In males at 1000 mg/kg/day a test item‑related increase in total T4 levels was recorded, with mean values on the upper limit of the historical control range. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity,grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption and clinical laboratory investigations (excluding male T4 thyroid hormone levels)).

In conclusion, based on the results of thiscombined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] was established:

Parental NOAEL for systemic effects: at least 1000 mg/kg/day

It is noted that in this study, a marked increase of total T4 was observed in high dose groups (in males) which was considered to be test item-related. However, under the conditions of this screening study no adverse effect was observed that could be linked to the increase of total T4 and therefore this increase was not taken into account when determining the parental NOAEL.

 .