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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In this OECD 422 study 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 were 0, 100, 300 and 1000 mg/kg/day.

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

The no-observed-adverse-effect levels (NOAEL) for reproduction was evaluated to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

NA

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (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.

Qualifier:

according to guideline

Guideline:

other: • EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.

Version / 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

Justification for study design:

NA

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)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat strain (Crl:WI(Han) rats) 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.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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

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. 20212886), and in an attempt to produce graded responses to the test item

Details on mating procedure:

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.

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

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. 20212886).

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 ± 10% 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. 20212886) 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 No20212888 .

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 avage 7 days a week for a minimum of 28 days.

Details on study schedule:

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.

Dose / conc.:

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

Remarks:

Vehicle (corn oil)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 male/10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. 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 assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Positive control:

No

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined included testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology (see attached study report).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- eight pups from each litter of equal sex distribution were 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 (see attachedd study report)

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25 (nos.65).
Without evidence of mating: 26 days after the last day of the mating period (no. 51).

All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were
stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

GROSS NECROPSY
- Full Gross necropsy was performed (See attached study report)

HISTOPATHOLOGY / ORGAN WEIGHTS
- A full histopathological evaluation was performed and organ weights collected in accordance to OECD 422 testing guideline requirements (see attached study report). Representative samples of tissues identified were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. All tissues as defined under Histology – F0-Generation (section 4.12.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist.

Organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16 PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
A full histopathological evaluation was perfomed and oragn weights noted in accordance to requirements in OECD 422 testing guideline (see attached study report).

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

Reproductive indices:

Mating index (%); Precoital time; Fertility index (%); Gestation index (%); Duration of gestation;

Offspring viability indices:

Post-implantation survival index (%); Live birth index (%); Percentage live males/females at First Litter Check (%), Viability index (%); Lactation index (%);

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:

no effects observed

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.

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.

Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for Female No. 55 at 100 mg/kg/day (with normal litter). In absence of a dose-related incidence and correlation to pregnancy status, this finding did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was 1/10 couples (male No. 11 and female No. 51) at 100 mg/kg/day and 1/10 couples (male No. 25 and female No. 65) at 300 mg/kg/day with no offspring (Text Table 17). No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Fertility index was considered not to be affected by treatment. The fertility indices were 100, 100, 90 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One female (No. 65) at 300 mg/kg/day was not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Number of implantation sites was considered not to be affected by treatment.

Precoital time was considered not to be affected by treatment. The majority of females showed evidence of mating within 5 days, except for one female each in the control and 1000 mg/kg/day groups for which it took 13 and 14 days, respectively, before mating could be confirmed. In the absence of a dose-related trend, it was considered unrelated to treatment.

Duration of gestation was considered not to be affected by treatment.
The number of females with living pups on lactation Day 1 compared to the number of pregnant females was decreased at 1000 mg/kg/day. The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 80% for the 1000 mg/kg/day group. The lower gestation index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74.

For developmental effects the following was concluded:

- Duration of gestation was considered not to be affected by treatment. The number of females with living pups on lactation Day 1 compared to the number of pregnant females was decreased at 1000 mg/kg/day. The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 80% for the 1000 mg/kg/day group. The lower gestation index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74.

- Female Nos. 72 and 74 at 1000 mg/kg/day were sacrificed on Day 23 post coitum with delivery difficulties. See section 9.2.1 for details. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (i.e. total number of offspring born as percentage of total number of uterine implantation sites) was 89, 90, 96 and 76% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The lower post-implantation survival index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74.

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 reproductive parameters affected

Critical effects observed:

no

Clinical signs:

not examined

Reproductive function: oestrous cycle:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of female No. 76 (1000 mg/kg/day) who was missing on Day 2, pale appearance and paresis were noted at first litter check.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Note: As female Nos. 72 and 74 at 1000 mg/kg/day were sacrificed in the post-coitum period, developmental data is available of only 8 females in the high dose group.

The number of females with living pups on lactation Day 1 compared to the number of pregnant females was decreased at 1000 mg/kg/day. The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 80% for the 1000 mg/kg/day group. The lower gestation index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74.

Live birth index (i.e. number of live offspring on PND 1 as percentage of total number of offspring born) was unaffected by treatment. The live birth index was 100% for all groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weights of pups were noted from Day 4 onwards, statistically significant on most occasions. As mean values remained within the historical control range , these changes were not considered adverse.

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:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In male PND 14-16 pups at 1000 mg/kg/day TSH levels were increased (1.24x of control, not statistically significant). Mean values were on the upper limit of the historical control range . Total T4 levels in PND 4 pups at 1000 mg/kg/day were decreased (0.69x of control, not statistically significant). Mean values were outside the historical control range . These changes were considered treatment related.

Serum levels of total T4 in male and female PND 14-16 pups and TSH levels in female PND 14-16 pups were considered not to be affected by treatment.

The statistically significantly increased decreased serum levels of total T4 in male PND 14-16 pups at 100 and 300 mg/kg/day were considered the result of relatively high control values, and therefore not regarded as toxicologically relevant. In absence of a dose-related trend, the increased TSH levels in female PND 14-16 pups at 300 mg/kg/day were considered not related to treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups.

Histopathological findings:

no effects observed

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Duration of gestation was considered not to be affected by treatment. The number of females with living pups on lactation Day 1 compared to the number of pregnant females was decreased at 1000 mg/kg/day. The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 80% for the 1000 mg/kg/day group. The lower gestation index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74 (for details see section 9.2.1 in attached testing report).

Female Nos. 72 and 74 at 1000 mg/kg/day were sacrificed on Day 23 post coitum with delivery difficulties. See section 9.2.1 for details.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index (i.e. total number of offspring born as percentage of total number of uterine implantation sites) was 89, 90, 96 and 76% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. The lower post-implantation survival index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74 (for details see section 9.2.1).

Litter size was considered not to be affected by treatment.
Live litter sizes were 9.3, 12.0, 10.8 and 12.5 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Live birth index (i.e. number of live offspring on PND 1 as percentage of total number of offspring born) was unaffected by treatment. The live birth index was 100% for all groups.

Viability index (i.e. number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 100, 100, 99 and 96% for the control, 100, 300 and 1000 mg/kg/day groups, respectively, which was within the historical control range. One pup at 300 and four pups at 1000 mg/kg/day were found missing between PND 2-4. These missing pups were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.


The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was unaffected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Base on delivery difficulties in 2/10 high dose females resulting in decreased gestation index and post implantation survival index

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

other: based on delivery difficulties in 2/10 females resulting in decreased gestation index and post implantation survival index

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

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 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.

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

No developmental toxicity was observed up to 300 mg/kg/day. Lower gestation index and post-implantation survival index were recorded at 1000 mg/kg/day (2/10). These changes were caused by the two females at 1000 mg/kg/day that were sacrificed with delivery difficulties on Day 23 post-coitum. As this phenomenon is uncommon, it is considered as possibly test item‑related.

At 1000 mg/kg/day increased TSH levels were recorded inmale PND 14-16 pups, while total T4 levels in PND 4 pups were decreased. These changes were considered treatment-related.

No adverse changes were noted in any of the other developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, sex ratio, maternal care, litter size, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retentionand macroscopic examination).

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 and developmental toxicity was evaluated to be 1000 mg/kg bw/day and 300 mg/kg bw/day, respectively.

In this study, a marked increase of total T4 and TSH 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 guideline and GLP study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In this OECD 422 study 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl (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 were 0, 100, 300 and 1000 mg/kg/day.

No developmental toxicity was observed up to 300 mg/kg/day. Lower gestation index and post-implantation survival index were recorded at 1000 mg/kg/day (2/10). These changes were caused by the two females at 1000 mg/kg/day that were sacrificed with delivery difficulties on Day 23 post-coitum. As this phenomenon is uncommon, it is considered as possibly test item‑related.

At 1000 mg/kg/day increased TSH levels were recorded inmale PND 14-16 pups, while total T4 levels in PND 4 pups were decreased. These changes were considered treatment-related.

No adverse changes were noted in any of the other developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, sex ratio, maternal care, litter size, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retentionand macroscopic examination).

The no-observed-adverse-effect levels (NOAEL) for developmental toxicity was evaluated to be 300 mg/kg bw/day.

In this study, a marked increase of total T4 and TSH 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 guideline and GLP study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

In this OECD 422 study 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl (PE120) when given orally by gavage for a minimum of 28 days to Wistar Han rats, using dose levels of 0, 100, 300 and 1000 mg/kg/day, no developmental toxicity was observed up to 300 mg/kg/day. Lower gestation index and post-implantation survival index were recorded at 1000 mg/kg/day (2/10). These changes were caused by the two females at 1000 mg/kg/day that were sacrificed with delivery difficulties on Day 23 post-coitum. As this phenomenon is uncommon, it is considered as possibly test item‑related.

No adverse changes were noted in any of the other developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, sex ratio, maternal care, litter size, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retentionand macroscopic examination).

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

At 1000 mg/kg/day increased TSH levels were recorded inmale PND 14-16 pups, while total T4 levels in PND 4 pups were decreased. These changes were considered treatment-related.

The no-observed-adverse-effect levels (NOAEL) for reproductivel toxicity was evaluated to be 300 mg/kg bw/day.

It is notedt that in this study, a marked increase of total T4 and TSH 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.

Based on these effects, no classification apply.