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REACH

Oxirane, mono[(C12-14-alkyloxy)methyl] derivs.

EC number: 271-846-8 | CAS number: 68609-97-2

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

An Extended One Generation Reproductive Toxicity Study is ongoing, and the present dossier will be updated with a robust study summary when available. 
Please see Section 7.8.1. 


Oxirane, 2-((C12-14-alkyloxy)methyl)derivs displayed no indications of any adverse effects on development in rats after dermal treatment at 1, 10, 50, 100, and 200 mg/kg/d from gestation days 6 to 15. The NOAELs for developmental toxicity and maternal systemic toxicity were 200 mg/kg bw/day. 
In the 13-week dermal study in Fisher rats, gross and histopathologic examinations revealed no adverse effects on the ovaries and testes of animals dosed with the substance at 100 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4420 (Preliminary Developmental Toxicity Screen)

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Sex:

female

Details on test animals or test system and environmental conditions:

Sixty sexually mature, virgin female Cr1:CD® (SD)BR rats were received in good health from Charles River Laboratories, Inc., Portage, Michigan, on October 17, 1996. The animals were approximately 10 weeks old. Upon receipt, each animal was observed by a qualified technician. The animals were initially weighed on the day following receipt. All animals were uniquely identified by a Monel metal eartag displaying the animal number and housed for 12 days for acclimation purposes. During this time, the animals were observed twice daily for mortality and moribundity.

ANIMAL HOUSING
Upon arrival and until pairing, all animals were individually housed in clean, wire-mesh cages suspended above cage-board. All animals were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH, 1985). The animal facilities at WIL Research Laboratories, Inc., are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

DIET. DRINKING WATER AND MAINTENANCE
The basal diet used in this study was PMI Feeds, Inc.© Certified Rodent LabDiet© 5002. This diet is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, Inc. Municipal water supplying the facility is sampled for contaminants according to Standard Operating Procedures. The results of these analyses are maintained at WIL Research Laboratories, Inc. Contaminants were not present in animal feed or water at concentrations expected to interfere with the objectives of this study. Drinking water delivered by an automatic watering system and the basal diet were provided ad libitum throughout the acclimation period and during the study.

F. ENVIRONMENTAL CONDITIONS
All animals were housed throughout the acclimation period and during the study in an environmentally-controlled room. Controls were set to maintain a temperature of 72° ± 4°F and a relative humidity of 30-70%. Room temperature and relative humidity were recorded daily. Actual recorded ranges for temperature and relative humidity were 71.6°F to 72.2°F and 26.4% to 45.5 %, respectively, during the study period. The occasional deviations from the set humidity levels would not be expected to have an adverse effect on the outcome of the study. Light timers were calibrated to provide a 12-hour light/12-hour dark photoperiod. Air handling units were set to provide approximately 10 fresh air changes per hour.

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
PREPARATION
A sufficient amount of the vehicle, acetone, was dispensed into a labeled storage container for administration to the control group. A magnetic stir bar was added and the solution was stirred continuously throughout the dispensing procedure. A sufficient amount of vehicle was dispensed into daily aliquots for dose administration.

Each test article formulation was prepared by weighing the appropriate amount of alkyl glycidyl ethers into a tared, precalibrated, labeled storage container and adding an appropriate amount of vehicle. A magnetic stir bar was added to each formulation, and the formulations were stirred continuously throughout the dispensing procedure. The preparations were dispensed into daily aliquots for dose administration. The test article
formulations were prepared once during the study (November 4, 1996) and were stored at room temperature, capped with nitrogen. The formulations visually inspected by the study director prior to the initiation of dosing on November 4, 1996, and were found to be acceptable for dose administration.

VEHICLE
The vehicle control article used for dose administration to the vehicle control group and in preparation of the test mixtures was acetone received from Fisher Scientific Company, Pittsburgh, Pennsylvania.

Details on mating procedure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

Gestation days 6 through 15.

Frequency of treatment:

six hours each day

Details on study schedule:

STUDY DESIGN
I. Acclimation Period
II. Untreated Females Paired with Untreated Resident Males
III. Animals Observed Daily, Body Weights and Food Consumption Recorded at Appropriate Intervals
IV. Mated Females/Group (1-6) Treated Once Daily (6 Hours/Day) From Gestation Days 6-15
V. Laparohysterectomies Performed on Gestation Day 20; Gravid Uterine Weights Recorded
VI. External Fetal Morphological Examination

Approximately 24 hours before the initial application of test article, the back (approximately 10% of the body surface) of each rat was clipped with Oster® small animal clippers. Clipping was repeated, if necessary, during the treatment period.

The test article, alkyl glycidyl ethers, was applied to the clipped intact dorsal skin of each test animal for six hours per day, from gestation days 6 through 15. The application sites were not occluded or abraded. Individual doses were calculated based on the most recent individual body weight recorded prior to test article application. All animals were dosed at approximately the same time each day.

The concurrent control group was dosed with the vehicle control article following procedures identical to those described for the treated groups.

During the exposure period, oral ingestion of the control and test articles was prevented by placing an Agar™ collar on each animal. Following the 6-hour exposure period, the collars were removed and the application sites were washed with a mild concentration of Ivory soap, then towel-dried. (The animals were acclimated to the collars prior to breeding according to the protocol. After breeding, the animals were fitted with collars for 6-hours per day throughout the pre-administration phase of gestation days 0-5 and during the treatment period. Acclimation to collars was suspended for each female upon pairing until confirmation of mating.)

The following diagram represents the study group assignment:
Group...............Dose Level.............Dose Concentration........Dose Volume.......Number of females
.......................... (mg/kg/day)................. (mg/ml)....................(ml/kg)
1........................0 (vehicle control).....................0.........................1........................8
2............................1................................................1........................1........................8
3..........................10.............................................10........................1........................8
4..........................50.............................................50........................1....................... 8
5........................100..........................................100........................1........................8
6........................200......................................... 200........................1........................8

No. of animals per sex per dose:

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily for moribundity and mortality. Individual detailed clinical observations were recorded from gestation days 0 through 20 (prior to compound administration during the dosing period). Clinical findings observed at the time of dosing, if present, were also recorded. All animals were observed for signs of toxicity approximately one hour following dosing; no significant findings were recorded at the post-dosing observation periods.
DERMAL IRRITATION
Application sites were examined for erythema, edema and other dermal findings once daily prior to treatment (gestation days 6 through 15) and daily thereafter. Erythema and edema were evaluated on a four step grading system of very slight, slight, moderate and severe. Other dermal findings, if present, were noted.

BODY WEIGHT: Yes
Individual maternal body weights were recorded on gestation days 0, 6-16 (daily) and 20. A group mean body weight was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-16, 6-16 and 0-20.
Gravid uterine weight was collected and net body weight (the day 20 body weight minus the weight of the uterus and contents) and net body weight change (the day 0-20 body weight change minus the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION: Yes
Individual maternal food consumption was recorded on the corresponding gestation body weight days. The amounts of food consumed were calculated for the corresponding body weight change intervals and were reported as g/animal/day and g/kg/day.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

Ovaries and uterine content
GESTATION DAY 20 LAPAROHYSTERECTOMY
All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed, opened and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski (1964, Archiv. Fur Exper. Pathologie und Pharm. 247:367. Maternal tissues were retained only as deemed necessary by the gross findings.

Litter observations:

The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed, opened and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski (1964, Archiv. Fur Exper. Pathologie und Pharm. 247:367. Maternal tissues were retained only as deemed necessary by the gross fmdings.

Postmortem examinations (parental animals):

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20

Ovaries and uterine content
GESTATION DAY 20 LAPAROHYSTERECTOMY
All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed, opened and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski (1964, Archiv. Fur Exper. Pathologie und Pharm. 247:367. Maternal tissues were retained only as deemed necessary by the gross fmdings.

Postmortem examinations (offspring):

FETAL MORPHOLOGICAL EXAMINATION
A detailed external examination of each fetus was conducted to include, but was not limited to, the eyes, palate and external orifices. Each fetus was then sexed, weighed, euthanized by an intrathoracic injection of pentobarbital and discarded. Findings were recorded as either developmental variations or malformations. Crown-rump measurements were recorded for late resorptions, if present, and the tissues were discarded.

Statistics:

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Dermal irritation (Erythema, edema and desquamation) at the application site was observed in the 50, 100 and 200 mg/kg/day groups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL OBSERVATIONS AND SURVIVAL
All animals survived to the scheduled necropsy on gestation day 20. In the 50, 100 and 200 mg/kg/day groups, four, five and six animals, respectively, were noted to vocalize at the time of dosing between gestation days 7 and 15. No other clinical signs that could be attributed to treatment with the test article were observed at any dose level. Other findings in the treated groups were limited to hair loss or scabbing on various body surfaces in single animals. No relationship to treatment was evident.

DERMAL IRRITATION
Erythema, edema and desquamation occurred in all animals in these groups (with the exception of no edema for one 50 mg/kg/day group animal). The severity and/or time of onset for these findings were dose-related. More severe signs of dermal irritation were noted only at dose levels of 100 and 200 mg/kg/day. These included fissuring, eschar formation and atonia in two to five 100 mg/kg/day group animals and in all 200 mg/kg/day group animals. The severity of erythema and edema and the frequency of fissuring, eschar formation and atonia tended to decrease during the post-treatment period.
Erythema was noted for all animals in the 50, 100 and 200 mg/kg/day groups beginning gestation days 7 or 8 (one to two days following the initiation of dosing) and persisting to study termination (gestation day 20). Erythema was graded as very slight to moderate in all three groups. Severe erythema was noted only at dose levels of 100 and 200 mg/kg/day beginning on gestation day 13 or 14 and continuing through gestation day 17 (100 mg/kg/day) or 19 (200 mg/kg/day). With the exception of female No. 57633 in the 50 mg/kg/day group, all animals in the 50, 100 and 200 mg/kg/day group had edema, generally graded as very slight in the 50 mg/kg/day group and as very slight to slight in the 100 and 200 mg/kg/day groups. Edema was first noted on gestation day 10 in the 50 mg/kg/day group and occurred sporadically thereafter. In the 100 and 200 mg/kg/day groups, edema was noted in all animals by gestation days 11 and 9, respectively, and generally persisted throughout the remainder of the treatment period and into the post-treatment period. Edema was not observed in either group on gestation day 20. Both erythema and edema tended to decrease in severity by the end of the post-treatment period (gestation day 20). Desquamation was observed in all animals in the 50, 100 and 200 mg/kg/day groups beginning gestation 12-16, 11-14 and 11-14, respectively. This finding persisted through gestation day 20.

Fissuring occurred in the 100 and 200 mg/kg/day groups beginning on gestation days 11 and 10, respectively. Eschar formation was first noted in these groups on gestation days 15 and 12, respectively. Atonia was first noted on gestation day 16 in the 100 mg/kg/day group and on gestation day 14 in the 200 mg/kg/day group. These findings were noted sporadically throughout the remainder of the study; however, the frequency of these findings tended to decrease during the post-treatment period (gestation days 16-20).

In the 10 mg/kg/day group, two females (nos. 57617 and 57655) had very slight erythema on gestation days 14 through 16. These transient occurrences in two animals were not considered to be definitive indications of test article- related dermal irritation. No other dermal findings were observed at a dose level of 10 mg/kg/day. No dermal findings were noted in the control or 1 mg/kg/day groups.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Mean body weights and body weight gains throughout gestation, net body weights, net body weight gains and gravid uterine weights in the 1, 10, 50, 100 and 200 mg/kg/day groups were similar to the control group values. Differences were slight and were not statistically significant.

FOOD CONSUMPTION
Food consumption, evaluated as g/animal/day and g/kg/day, in the 1, 10, 50, 100 and 200 mg/kg/day groups was unaffected by test article administration. The only statistically significant difference between the control and treated groups during the comprehensive intervals was a slightly decreased (p <0.05) g/kg/day food consumption value in the 1 mg/kg/day group during gestation days 12-16. Similar decreases were not observed at the higher dose levels, and no relationship to treatment was apparent.

NECROPSY DATA
At the scheduled necropsy, test article-related findings were limited to observations at the application site. Two and six animals in the 100 and 200 mg/kg/day groups, respectively, had scabbing at the application site. Two of these 200 mg/kg/day group animals also had thickening at the application site. No internal findings related to the test article were observed at any dose level. Female no. 57658 in the 200 mg/kg/day group had dark red lungs. All other animals were internally normal.

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
Intrauterine growth and survival were unaffected by test article administration at any dose level. Parameters evaluated included postimplantation loss, mean fetal body weights, viable litter size, fetal sex ratios and the mean numbers of corpora lutea and implantation sites. None of the differences from the control group were statistically significant.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No maternal toxicity or developmental toxicity was apparent at any dose level.

Dose descriptor:

NOEL

Remarks:

dermal irritation

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Dermal irritation (Erythema, edema and desquamation) at the application site was observed in the 50, 100 and 200 mg/kg/day groups.

Critical effects observed:

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

The numbers of fetuses (litters) available for external morphological evaluation were 99(7), 110(8),91(7), 79(7), 95(7) and 87(6) in the control, 1, 10, 50, 100 and 200 mg/kg/day groups, respectively. Control group fetus no. 57646-05 had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: One control group fetus had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

Critical effects observed:

Reproductive effects observed:

not specified

Conclusions:

In conclusion, localized dermal irritation was observed in a dose-related manner at dose levels of 50, 100 and 200 mg/kg/day. No maternal toxicity or developmental toxicity was apparent at any dose level. Based on the results of this study, the no-effect level for dermal irritation was considered to be 10 mg/kg/day and the no-effect level for maternal toxicity and developmental toxicity was considered to be 200 mg/kg/day.

Executive summary:

All maternal animals survived to the scheduled necropsy on gestation day 20.

The only treatment-related clinical sign observed was vocalization at the time of dosing in the 50, 100 and 200 mg/kg/day groups between gestation days 7 and 15.

Dermal irritation at the application site was observed in the 50, 100 and 200 mg/kg/day groups. Erythema, edema and desquamation occurred in all animals in these groups (with the exception of no edema for one 50 mg/kg/day group animal). The severity and/or time of onset for these findings were dose-related. More severe signs of dermal irritation were noted only at dose levels of 100 and 200 mg/kg/day. These included fissuring, eschar formation and atonia in two to five 100 mg/kg/day group animals and in all 200 mg/kg/day group animals. The severity of erythema and edema and the frequency of fissuring, eschar formation and atonia tended to decrease during the post-treatment period. No test article-related dermal irritation was noted at the application site in the 1

and 10 mg/kg/day groups.

Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 1, 10, 50, 100 and 200 mg/kg/day groups were similar to the control group values. Food consumption, assessed as g/animal/day and g/kg/day, in the treated groups was unaffected by test article administration.

At the scheduled necropsy, no test article-related internal findings were observed at any dose level. Findings at the application site were limited to scabbing in two and six animals in the 100 and 200 mg/kg/day groups, respectively, and thickening in two of these high dose group animals.

Intrauterine growth and survival were unaffected by test article administration at any dose level. Parameters evaluated included postimplantation loss, mean fetal body weight, viable litter size, fetal sex ratios and the mean numbers of corpora. lutea and implantation sites. Fetuses (litters) available for external morphological evaluation numbered 99(7), 110(8), 91(7), 79(7), 95(7) and 87(6) in the control, 1, 10, 50, 100 and 200 mg/kg/day groups, respectively. One control group fetus had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

Effect on fertility: via oral route

Endpoint conclusion:: no study available

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 100 mg/kg bw/day
Study duration:: subchronic
Species:: rat

Additional information

Justification for selection of Effect on fertility via dermal route:
In the 13-week dermal study in Fisher rats, gross and histopathologic examinations revealed no adverse effects on the ovaries and testes of animals dosed with the substance at 100 mg/kg/day.

Effects on developmental toxicity

Description of key information

In a GLP compliant, OECD 414 study conducted using Sprague Dawley rats, Oxirane, mono[(C12-14-alkyloxy)methyl] derivs was well tolerated and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no adverse effect on embryo-fetal survival, development or growth, therefore the fetal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day.

In a GLP compliant, OECD 414 study conducted using New Zealand White Rabbits, treatment was generally well-tolerated with no test item-related deaths. The No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and for embryo-fetal survival and development was concluded to be 375 mg/kg/day.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Details on test animals or test system and environmental conditions:

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 3-ml samples were collected from the middle stratum of each dosing preparation, including the control, on the first day of dosing (November 4, 1996). One set of samples was analyzed for concentration. The remaining set was stored at room temperature, under nitrogen, then analyzed for stability after the last day of dosing. Due to the limited supply of test article and the small volumes formulated, samples for analysis were not collected as described in the protocol (one 10-ml sample from each formulation on the first and last days of dosing). This deviation would not be expected to have an adverse effect on the outcome of the study. All analyses were conducted by the Analytical Chemistry Department at WIL Research Laboratories, Inc. The methods and results of these analyses are presented in the study report. The dosing formulations contained the amounts of test article specified in the protocol and were stable for 12 days.

Details on mating procedure:

At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the study director, animals judged to be in good health and meeting acceptable body weight requirements (a minimum of 220 g at the initiation of mating) were placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was prepared. The selected females were approximately 12 weeks old when paired for breeding.

Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Each mating pair was examined daily. The day on which evidence of mating was identified was termed day 0 of gestation and the animals were separated.

Duration of treatment / exposure:

Gestation days 6 through 15.

Frequency of treatment:

A single daily application to six groups of eight bred Crl:CD@(SD)BR rats, from gestation
days 6 through 15.

Duration of test:

six hours each day

No. of animals per sex per dose:

Details on study design:

Maternal examinations:

Ovaries and uterine content:

GESTATION DAY 20 LAPAROHYSTERECTOMY
All maternal animals were euthanized by carbon dioxide inhalation on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed, opened and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Uteri with no macroscopic evidence of nidation were excised, opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss as described by Salewski (1964, Archiv. Fur Exper. Pathologie und Pharm. 247:367. Maternal tissues were retained only as deemed necessary by the gross fmdings.

Fetal examinations:

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each treated group to the vehicle control group. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. The following statistical tests were performed by a Digital® MicroVAX® 3400 computer (with appropriate programming) in this laboratory and are referenced on the report tables:

STATISTICAL TEST
PARAMETER

Chi-square test with Yates' correction factor:
Fetal Sex Ratios

Mann-Whitney U-test:
Early and Late Resorptions,
Dead Fetuses
Postimplantation Losses
ANOVA (two-tailed) with Dunnett's test
Corpora Lutea
Total Implantations
Viable Fetuses
Fetal Body Weights
Maternal Body Weights and Weight Changes
Maternal Net Body Weight Changes and Gravid UterineWeights
Maternal FoodConsumption

Indices:

Indices:
Group Mean Litter Basis:
Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group (divided by) No. Gravid Females/Group

Proportional Litter Basis:
Summation per Group (%) = Postimplantation Loss/Litter (%)a (divided by) No. of Litters/Group

a = No. Dead Fetuses, Resorptions (Early/Late)/Group x 100 (divided by) No. Implantation Sites/Litter

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

NOEL (dermal irritation) (P): 10 mg/kg bw/day (female) based on: test mat. (Dermal irritation (Erythema, edema and desquamation) at the application site was observed in the 50, 100 and 200 mg/kg/day groups.)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Number of abortions:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS AND SURVIVAL
All animals survived to the scheduled necropsy on gestation day 20. In the 50, 100 and 200 mg/kg/day groups, four, five and six animals, respectively, were noted to vocalize at the time of dosing between gestation days 7 and 15. No other clinical signs that could be attributed to treatment with the test article were observed at any dose level. Other findings in the treated groups were limited to hair loss or scabbing on various body surfaces in single animals. No relationship to treatment was evident.

DERMAL IRRITATION
Erythema, edema and desquamation occurred in all animals in these groups (with the exception of no edema for one 50 mg/kg/day group animal). The severity and/or time of onset for these findings were dose-related. More severe signs of dermal irritation were noted only at dose levels of 100 and 200 mg/kg/day. These included fissuring, eschar formation and atonia in two to five 100 mg/kg/day group animals and in all 200 mg/kg/day group animals. The severity of erythema and edema and the frequency of fissuring, eschar formation and atonia tended to decrease during the post-treatment period.
Erythema was noted for all animals in the 50, 100 and 200 mg/kg/day groups beginning gestation days 7 or 8 (one to two days following the initiation of dosing) and persisting to study termination (gestation day 20). Erythema was graded as very slight to moderate in all three groups. Severe erythema was noted only at dose levels of 100 and 200 mg/kg/day beginning on gestation day 13 or 14 and continuing through gestation day 17 (100 mg/kg/day) or 19 (200 mg/kg/day). With the exception of female No. 57633 in the 50 mg/kg/day group, all animals in the 50, 100 and 200 mg/kg/day group had edema, generally graded as very slight in the 50 mg/kg/day group and as very slight to slight in the 100 and 200 mg/kg/day groups. Edema was first noted on gestation day 10 in the 50 mg/kg/day group and occurred sporadically thereafter. In the 100 and 200 mg/kg/day groups, edema was noted in all animals by gestation days 11 and 9, respectively, and generally persisted throughout the remainder of the treatment period and into the post-treatment period. Edema was not observed in either group on gestation day 20. Both erythema and edema tended to decrease in severity by the end of the post-treatment period (gestation day 20). Desquamation was observed in all animals in the 50, 100 and 200 mg/kg/day groups beginning gestation 12-16, 11-14 and 11-14, respectively. This finding persisted through gestation day 20.

Fissuring occurred in the 100 and 200 mg/kg/day groups beginning on gestation days 11 and 10, respectively. Eschar formation was first noted in these groups on gestation days 15 and 12, respectively. Atonia was first noted on gestation day 16 in the 100 mg/kg/day group and on gestation day 14 in the 200 mg/kg/day group. These findings were noted sporadically throughout the remainder of the study; however, the frequency of these findings tended to decrease during the post-treatment period (gestation days 16-20).

In the 10 mg/kg/day group, two females (nos. 57617 and 57655) had very slight erythema on gestation days 14 through 16. These transient occurrences in two animals were not considered to be definitive indications of test article- related dermal irritation. No other dermal findings were observed at a dose level of 10 mg/kg/day. No dermal findings were noted in the control or 1 mg/kg/day groups.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
Mean body weights and body weight gains throughout gestation, net body weights, net body weight gains and gravid uterine weights in the 1, 10, 50, 100 and 200 mg/kg/day groups were similar to the control group values. Differences were slight and were not statistically significant.

FOOD CONSUMPTION
Food consumption, evaluated as g/animal/day and g/kg/day, in the 1, 10, 50, 100 and 200 mg/kg/day groups was unaffected by test article administration. The only statistically significant difference between the control and treated groups during the comprehensive intervals was a slightly decreased (p <0.05) g/kg/day food consumption value in the 1 mg/kg/day group during gestation days 12-16. Similar decreases were not observed at the higher dose levels, and no relationship to treatment was apparent.

NECROPSY DATA
At the scheduled necropsy, test article-related findings were limited to observations at the application site. Two and six animals in the 100 and 200 mg/kg/day groups, respectively, had scabbing at the application site. Two of these 200 mg/kg/day group animals also had thickening at the application site. No internal findings related to the test article were observed at any dose level. Female no. 57658 in the 200 mg/kg/day group had dark red lungs. All other animals were internally normal.

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
Intrauterine growth and survival were unaffected by test article administration at any dose level. Parameters evaluated included postimplantation loss, mean fetal body weights, viable litter size, fetal sex ratios and the mean numbers of corpora lutea and implantation sites. None of the differences from the control group were statistically significant.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for external morphological evaluation were 99(7), 110(8),91(7), 79(7), 95(7) and 87(6) in the control, 1, 10, 50, 100 and 200 mg/kg/day groups, respectively. Control group fetus no. 57646-05 had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

All maternal animals survived to the scheduled necropsy on gestation day 20.
The only treatment-related clinical sign observed was vocalization at the time of dosing in the 50, 100 and 200 mg/kg/day groups between gestation days 7 and 15.

Dermal irritation at the application site was observed in the 50, 100 and 200 mg/kg/day groups. Erythema, edema and desquamation occurred in all animals in these groups (with the exception of no edema for one 50 mg/kg/day group animal). The severity and/or time of onset for these findings were dose-related. More severe signs of dermal irritation were noted only at dose levels of 100 and 200 mg/kg/day. These included fissuring, eschar formation and atonia in two to five 100 mg/kg/day group animals and in all 200 mg/kg/day group animals. The severity of erythema and edema and the frequency of fissuring, eschar formation and atonia tended to decrease during the post-treatment period. No test article-related dermal irritation was noted at the application site in the 1
and 10 mg/kg/day groups.

Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 1, 10, 50, 100 and 200 mg/kg/day groups were similar to the control group values. Food consumption, assessed as g/animal/day and g/kg/day, in the treated groups was unaffected by test article administration.

At the scheduled necropsy, no test article-related internal findings were observed at any dose level. Findings at the application site were limited to scabbing in two and six animals in the 100 and 200 mg/kg/day groups, respectively, and thickening in two of these high dose group animals.

Intrauterine growth and survival were unaffected by test article administration at any dose level. Parameters evaluated included postimplantation loss, mean fetal body weight, viable litter size, fetal sex ratios and the mean numbers of corpora. lutea and implantation sites. Fetuses (litters) available for external morphological evaluation numbered 99(7), 110(8), 91(7), 79(7), 95(7) and 87(6) in the control, 1, 10, 50, 100 and 200 mg/kg/day groups, respectively. One control group fetus had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

In conclusion, localized dermal irritation was observed in a dose-related manner at dose levels of 50, 100 and 200 mg/kg/day. No maternal toxicity or developmental toxicity was apparent at any dose level. Based on the results of this study, the no-effect level for dermal irritation was considered to be 10 mg/kg/day and the no-effect level for maternal toxicity and developmental toxicity was considered to be 200 mg/kg/day.

Executive summary:

The potential maternal toxicity and developmental toxicity of alkyl glycidyl ethers were evaluated. The test article in acetone was administered as a single daily application to six groups of eight bred Crl:CD@(SD)BR rats, from gestation days 6 through 15. The test article was applied to the clipped intact dorsal skin (10% area) of each rat. The application sites were not occluded. Dosage levels were 1, 10, 50, 100 and 200 mg/kg/day administered at a dose volume of 1 ml/kg. A concurrent control group, composed of eight bred females, received the vehicle, acetone, on a comparable regimen at 1 ml/kg. All animals were fitted with Agar™ collars six hours each day (on the days prior to treatment and during the exposure period) to minimize oral ingestion of the vehicle or test article. Throughout gestation all females were observed twice daily for mortality and moribundity. Detailed clinical observations were recorded once daily. Dermal observations were recorded daily (gestation days 6-20) prior to administration. Body weights and food consumption were recorded at appropriate intervals. On day 20 of gestation, all females were euthanized and scheduled laparohysterectomies were performed. The uteri and ovaries were examined and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Mean gravid uterine weights and net body weight changes were calculated for each group. Fetuses were weighed, sexed and examined for external malformations and developmental variations.

All maternal animals survived to the scheduled necropsy on gestation day 20; no test article-related internal findings were observed. The only treatment-related clinical sign observed was vocalization at the time of dosing in the 50, 100 and 200 mg/kg/day groups. Body weight gain and food consumption in the treated groups were unaffected by test article administration. Intrauterine growth and survival were also unaffected at all dose levels. No external fetal malformations were observed in the treated groups, and no external developmental variations were noted in fetuses in this study.

Dermal irritation at the application site was observed in the 50, 100 and 200 mg/kg/day groups. Erythema, edema and desquamation occurred in all animals in these groups (with the exception of no edema for one 50 mg/kg/day group animal). The severity and/or time of onset for these findings were dose-related. More severe signs of dermal irritation were noted only at dose levels of 100 and 200 mg/kg/day. These included fissuring, eschar formation and atonia in100 mg/kg/day group animals and in all 200 mg/kg/day group animals. The severity of erythema and edema and the frequency of fissuring, eschar formation and atonia tended to decrease during the post-treatment period. No test article-related dermal irritation was noted at the application site in the 1 and 10 mg/kg/day groups. At necropsy, findings at the application site included scabb

ing in two 100 mg/kg/day group animals and scabbing and/or thickening in six 200 mg/kg/day group animals.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Pre-study chemistry) 17 May 2018

Experimental completion date (fetal evaluation) :29 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: JMAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

Appearance: Clear liquid.
Storage conditions: Ambient (15 to 25°C), in the dark.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: 18 to 22 weeks old
- Weight at study initiation: 2.50 to 4.44 kg
- Fasting period before study:
- Housing: One female per cage
- Diet (e.g. ad libitum): Teklad 2930 Diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Availability was Restricted (200 g/animal/day).
- Water: ad libitum
- Acclimation period: Five days from arrival on GD1 to start of treatment on GD6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C
- Humidity (%): 45-70%.
- Air changes : Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light : 10 hours dark

IN-LIFE DATES:
From: 10 February 2019
To: 29 March 2019

Route of administration:

oral: gavage

Vehicle:

other: aqueous 1% w/v methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Method of preparation
The required amount of test item was added to approximately 40% of the final volume. The formulation was magnetically stirred until uniformly mixed and then made to the required volume using the vehicle. It was finally mixed using a high-shear homogenizer.

A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Weekly.

Storage of formulation Refrigerated 2 to 8°C.

Test item accounting Detailed records of compound usage were maintained.

The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: Refrigerated 2 to 8°C

VEHICLE
- Concentration in vehicle: 40, 125, 375 mg/kg/day
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Purity: 1% w/v

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity Before commencement of treatment, the suitability of the
proposed mixing procedures was determined and specimen formulations at 1and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations were confirmed to be stable for one day at ambient temperature (15 to 25C) and for eight days after refrigerated storage (2 to 8C).

The analytical procedure was successfully validated for Oxirane, mono[(C12-14- alkyloxy)methyl] derivs: in 1% Methylcellulose with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision, calibration standard stability and extract stability.
The specificity of the GC assay was demonstrated by the absence of a peak at the characteristic retention time for Oxirane, mono[(C12-14-alkyloxy)methyl] derivs: in the control sample chromatogram.

The limit of detection and quantification was estimated as 0.225 µg/mL and 0.750 µg/mL respectively.

Linearity was confirmed over the nominal concentration range 10 µg/mL to 100 µg/mL with a coefficient of determination >0.998.

The repeatability was <2% for six replicate injections of standard solutions containing Oxirane, mono[(C12-14-alkyloxy)methyl] derivs: at nominal concentrations of 10 µg/mL and 100 µg/mL.

Method accuracy and precision were confirmed: a mean procedural recovery value of 104.9% (CV=1.27%, n=5) was obtained for 1 mg/mL and 100.6% (CV=1.15%, n=5) was obtained for 200 mg/mL. Calibration standard stability was confirmed after refrigerated storage for 4 days. Extract stability was confirmed after refrigerated storage for 4 days for 200 mg/mL samples only. The 1mg/mL samples were shown not to be stable after 4 days of refrigerated storage.

Details on mating procedure:

Natural mating with New Zealand white bucks of established fertility at the supplier’s facility. Males and females not closely related. After mating each female injected intravenously with 25 i.u. luteinising hormone.

Duration of treatment / exposure:

Females - Day 6 to 28 after mating

Frequency of treatment:

Daily

Duration of test:

22 days

Dose / conc.:

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

Remarks:

Control group

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
The doses used in this study (0, 40, 125 and 375 mg/kg/day) were selected based on on a rabbit oral gavage pilot study (Envigo study number HM82WV):

Rationale for Dose Level Selection

The doses used in this study (0, 40, 125 and 375 mg/kg/day) were selected in conjunction with the Sponsor.
On a rabbit oral gavage pilot study (Envigo study number HM82WV) dose levels of 250, 375 and 500 mg/kg/day were administered over a two week period.
500 mg/kg/day: reduced food, hay and water consumption, reduced faeces (faecal pellet size and number of pellets), yellow fur staining, body weight loss and reduced food consumption (at a level that could induce abortion in a pregnant animal) were evident following administration of 500 mg/kg/day.

375 mg/kg/day: little diet and/or hay eaten were evident in two animals and decreased
faeces, thin build and irritability were apparent in individual animals following administration of 375 mg/kg/day. Bodyweight stasis or slight body weight loss and reduced food consumption were also apparent.

250 mg/kg/day: little diet and/or hay eaten, decreased faeces, yellow fur staining, bodyweight stasis or slight body weight loss and reduced food consumption were evident following administration of 250 mg/kg/day.

No macroscopic abnormality was evident in any animal at any dose level.

On a preliminary embryo fetal study with pregnant animals (Envigo study DB39CF), dose levels of 90, 180 and 375 mg/kg/day were administered from the sixth day of gestation (GD6) until the 29th day (GD29). Treatment related findings were restricted to body weight loss during the first week of treatment in all test groups resulting in a dose-related lower than

control overall body weight gain and adjusted body weight change and reduced food intake in animals treated at 375 mg/kg/day during the second week of treatment.
For this main embryo fetal study with pregnant animals, dose levels of 40, 125 and 375 mg/kg/day were selected.

Maternal examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

CAGE SIDE OBSERVATIONS:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at
least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

One to two hours after completion of dosing. As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 1 (on arrival), 3, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT:
The weight of each adult was recorded on the day of arrival (Day 1 after mating) and on Days 3 and 6-29 after mating.

FOOD CONSUMPTION
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating.

POST-MORTEM EXAMINATIONS:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.

Sequence To allow satisfactory inter-group comparison.

Terminal Investigations Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) /Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses) /Number of implantations ) x 100

All group values and SD (as appropriate) were calculated from the individual litter values.

Ovaries and uterine content:

For females surviving to term, the following was recorded:
Uterus - Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn - Number of: corpora lutea, Implantation sites, Resorption sites (assessed as early or late), Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns - The absence or number of uterine implantation sites was confirmed.

Apparently non-pregnant animals and for apparently empty uterine horns

The absence or number of uterine implantation sites was confirmed.

Fetal examinations:

Examination of all viable fetuses and placenta:
Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation:
Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.
Remaining fetuses and torsos were eviscerated and fixed in Industrial Methylated Spirit.

Processing:
Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination

Bouin’s fixed heads:
Serial sections were examined for soft tissue abnormalities.

Alizarin Red stained fetuses and torsos:
Assessed for skeletal development and abnormalities.

Fetal, Litter and Placental Weights

Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination

Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:

Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. partially open eyelids, absent kidney/ureter.

Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.

Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs and thoracolumbar vertebra, incomplete ossification of 5th and 6th sternebrae.

In the Fetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistics:

Please refer to "ANY OTHER INFORMATION ON MATERIALS AND METHODS"

Indices:

Not stated

Historical control data:

PLEASE REFER TO THE ATTACHED TABLES: "HISTORICAL CONTROL DATA"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of little hay eaten, decreased fecal pellets and fecal pellets reduced in size were seen in a higher number of females receiving the test item at 375 mg/kg/day. This is likely to be related to treatment with the test item.

There were no other clinical signs or signs related to dosing which were considered related to treatment with the test item at 40, 125 or 375 mg/kg/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (4F 81) receiving 375 mg/kg/day was killed for welfare reasons on Day 27 of gestation with red perigenital staining and red staining on the tray paper and tray, whole body pallor and general poor clinical condition. This female was found to be pregnant with 9 live young and two late resorptions. Macroscopic examination at necropsy revealed dark fluid in the left uterine horn. Since this was an isolated occurrence, this death was not related to the administration of the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body Weight and Gravid Uterine Weight
There was no clear effect of treatment on the bodyweight performance of females receiving the test item at 40, 125 or 375 mg/kg/day.

The weight of the gravid uterus was higher than that of the control for females which received the test item at 40, 125 or 375 mg/kg/day. This is likely to be a result of the slightly higher number of fetuses in these groups which has arisen by chance and is unlikely to be related to treatment with the test item. When mean values of body weight and body weight gain were adjusted for the contributions of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was similar across all groups.

PLEASE REFER TO THE ATTACHED TABLE: "BODY WEIGHT AND BODY WEIGHT CHANGE - GROUP MEAN VALUES DURING GESTATION"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was no clear effect of treatment on the food consumption of females receiving the test item at 40 or 125 mg/kg/day.

At 375 mg/kg/day, mean food intake was marginally low on Days 17-23 of gestation and the differences attained statistical significance on Days 17-18, 20-21 and 22-23 of gestation.

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Treatment with test item was generally well-tolerated with no test item-related deaths.

There were no consistent adverse effect on the clinical signs, body weight or food consumption of the adult females. At 375 mg/kg/day, mean food intake was marginally low on Days 17-23 of gestation and the differences attained statistical significance on Days 17-18, 20-21 and 22-23 of gestation.

The gravid uterine weight, embryo-fetal survival and development and fetal pathology were not adversely affected by the test item in all treatment groups.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Embryo-fetal survival was unaffected across all treatment groups as assessed by mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and post-implantation loss.

PLEASE REFER TO THE ATTACHED TABLE:"LITTER DATA - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Early or late resorptions:

no effects observed

Description (incidence and severity):

Embryo-fetal survival was unaffected across all treatment groups as assessed by mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre- and post-implantation loss.
PLEASE REFER TO THE ATTACHED TABLE:"LITTER DATA - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Dead fetuses:

no effects observed

Description (incidence and severity):

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Other effects:

not examined

Details on maternal toxic effects:

In addition to the premature death observed due to animal welfare reasons in the
375 mg/kg/day group, there were also three non-pregnant animals in the control group,
five non-pregnant animals in the treatment group receiving 40 mg/kg/day, one non-pregnant animal in the treatment group receiving 125 mg/kg/day, and five non-pregnant animals in the treatment group receiving 375 mg/kg/day. The litter data assessment is therefore based on a total of 21, 19, 23 and 18 pregnant females reaching gestation Day 29 in each of the Groups 1, 2, 3, 4, respectively.

At 125 and 375 mg/kg/day there was a slight increase in delayed ossification of the metacarpals and phalanges compared to concurrent control but within Historical Control Data range and so no effect of treatment is inferred. As this is a transient stage of fetal development, it is not considered adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

mortality

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean placental and overall fetal weights were similar to the Control group and not affected by treatment with the test item. The mean total litter weight at 40, 125 and 375 mg/kg/day was higher than that of the Control due to the slightly higher number of fetuses in these groups; no effect of treatment is inferred.

PLEASE REFER TO THE FOLLOWING ATATCHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE ATTACHED TABLE:"LITTER DATA - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Changes in sex ratio:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE ATTACHED TABLE:"LITTER DATA - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE ATTACHED TABLE:"LITTER DATA - GROUP MEAN VALUES ON DAY 29 OF GESTATION"

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no major abnormalities considered to be related to treatment.

At 125 and 375 mg/kg/day there was a slight increase in delayed ossification of the metacarpals and phalanges compared to concurrent control but within Historical Control Data range and so no effect of treatment is inferred. As this is a transient stage of fetal development, it is not considered adverse.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES:
1) FETAL EXAMINATIONS_MAJOR ABNORMALITY FINDINGS - GROUP INDICES
2) FETAL EXAMINATIONS - MINOR SKELETAL ABNORMALITY FINDINGS - GROUP INCIDENCES

Visceral malformations:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE FLLOWING ATTACHED TABLE: "FETAL EXAMINATIONS_MINOR VISCERAL ABNORMALITY AND NECROPSY FINDINGS - GROUP INDICES"

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

Lowest effective dose / conc.:

375 mg/kg bw/day (nominal)

Treatment related:

yes

Formulation Analysis

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision, calibration standard stability and high level extract stability. The low level extract stability was not confirmed after 4 days at refrigerated storage.

The homogeneity and stability was confirmed for Oxirane, mono[(C12-14-alkyloxy)methyl] derivs: in 1% Methylcellulose formulations at nominal concentrations of 1 mg/mL and

200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 8 days.

The mean concentrations of Oxirane, mono[(C12-14-alkyloxy)methyl] derivs: in test formulations analyzed for the study were within ±5% of nominal concentrations, confirming accurate formulation. The difference from mean remained within 4%, confirming precise analysis.

Procedural recoveries remained within the validated range, confirming continued accuracy of the analytical method, with the exception of one recovery prepared during the first week of dosing, which has been excluded as an outlier as per SOP.

Conclusions:

Based on the results obtained in this main rabbit embryo-fetal development study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and for embryo-fetal survival and development was concluded to be 375 mg/kg/day.

Executive summary:

SUMMARY

The purpose of this study was the assessment of the influence of Oxirane, mono[(C12-14- alkyloxy)methyl] derivs, an industrial chemical, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phase of pregnancy in the New Zealand White rabbit.

Three groups of 24 females received Oxirane, mono[(C12-14-alkyloxy)methyl] derivs at doses of 40, 125 or 375 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, aqueous 1% w/v methylcellulose at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Results

The homogeneity and stability was confirmed for Oxirane, mono[(C12-14-alkyloxy)methyl] derivs: in 1% Methylcellulose formulations at nominal concentrations of 1 mg/mL and

200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 8 days.

One female (4F 81) receiving 375 mg/kg/day was killed for welfare reasons on Day 27 of gestation with red perigenital staining and red staining on the tray paper and tray, whole body pallor and general poor clinical condition. This female was found to be pregnant with 9 live young and two late resorptions. Macroscopic examination at necropsy revealed dark fluid in the left uterine horn. Since this is an isolated occurrence, this death is not related to the administration of the test item.

There were no other clinical signs or signs related to dosing which were considered related to treatment with the test item at 40, 125 or 375 mg/kg/day.

There was no clear effect of treatment on the bodyweight performance or food consumption of females receiving the test item at 40, 125 or 375 mg/kg/day.

The weight of the gravid uterus was higher than that of the control for females which received the test item at 40, 125 or 375 mg/kg/day. This is likely to be a result of the slightly higher number of fetuses in these groups which has arisen by chance and is unlikely to be related to treatment with the test item. When mean values of body weight and body weight gain were adjusted for the contributions of the gravid uterus, overall maternal mean body weight loss during Days 6-29 of gestation was similar across all groups.

There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 of gestation.

Embryo-fetal survival was unaffected by treatment at 40, 125 or 375 mg/kg/day as assessed by mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss.

Mean litter weights at 40, 125 and 375 mg/kg/day were marginally lower than Controls; this is likely to be a result of the marginally higher litter size in these groups which has occurred by chance.

Mean placental, male, female and overall fetal weights at 40, 125 or 375 mg/kg/day were similar to Controls and unaffected by treatment.

The incidence of major and minor abnormalities and skeletal variants at fetal pathology examination showed no relationship to treatment.

Conclusion

Based on the results obtained in this main rabbit embryo-fetal development study, the

No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and for embryo-fetal survival and development was concluded to be 375 mg/kg/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2018 - 02 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Storage conditions: Ambient (15 to 25°C), in the dark.
Batch number: AAF1453400.
Purity: UVCB

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 72 days old.
- Weight at study initiation: 234 to 290 g.
- Fasting period before study: not specified
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Non-restricted.

- Water (e.g. ad libitum):Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted.

- Acclimation period: Six days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, approximately 40% of the final volume of vehicle was added to the required amount of test item. This was magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order.

VEHICLE - Corn oil
- Justification for use and choice of vehicle (if other than water): According to guidelines / Homogeneity and stability of the test item
- Concentration in vehicle: 0 - 200 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.
The first and last formulations were sampled. For all groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation.
Two samples were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
For each occasion, procedural recoveries were prepared to cover the range of inclusions levels examined and analyzed concurrently with the test formulations as a quality control measure.

Stability and homogeneity Homogeneity and stability of the test item in the vehicle
was determined as part of this programme of work in Envigo Study No. RX82TW. Formulations from
1 mg/mL to 200 mg/mL are stable in corn oil for up to 15 days when stored refrigerated (2 to 8°C) and one day when stored at ambient temperature (15 to 25°C).
Achieved concentration Samples of each of the first and last formulations prepared
were analyzed for achieved concentration of the test item.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: Daily checks for evidence of mating
- M/F ratio per cage: 1:1
- Length of cohabitation: Until positive evidence of mating was detected.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Any other deviations from standard protocol: none

Duration of treatment / exposure:

14 days (Day 6 to 19 after mating)

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Duration of test:

25 April 2018 - 02 July 2018

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

330 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:

20 males, 20 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 100, 330 and 1000 mg/kg/day) were selected in conjunction with the Sponsor. Dose levels were selected based on the effects of a preliminary embryo-fetal study at this laboratory (Envigo Study Number TH72CQ) which investigated dose levels of 250, 500 and 1000 mg/kg/day. At 1000 mg/kg/day, overall body weight gain and food consumption were marginally low but there was no observed effect on fetal growth, development or survival. Therefore, dose levels of 100, 330 and 1000 mg/kg/day were selected for this main embryo-fetal study.
- Rationale for animal assignment (if not random): random

Maternal examinations:

Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing - Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

- Pre-dose observation
- One to two hours after completion of dosing
-As late as possible in the working day

Clinical Signs - A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight:
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Group mean weight changes were calculated from the weight changes of individual animals. Body weights were plotted graphically with respect to Day 6 of gestation.
Adjusted body weights on Day 20 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-20 were also presented, after being adjusted for the contribution of the gravid uterus.

Food Consumption:
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Necropsy:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule - Animals were killed on Day 20 after mating.

Sequence - To allow satisfactory inter-group comparison.

Ovaries and uterine content:

Reproductive Assessment - The following were recorded for all animals:
Uterus - Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn: Number of Corpora lutea, Implantation sites, Resorption sites (classified as early or late), and Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns - The number of uterine implantation sites were checked after staining with ammonium sulphide.

Fetal examinations:

Fetal Examination and Processing:

Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter - Sexed internally and eviscerated.
Fixation - Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing - Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and stained with Alizarin
Red.

Fetal, Litter and Placental Weights

Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination

Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:

Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.

Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.

Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

In the Fetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Fetal Pathology Examination:
Bouin’s fixed fetuses - Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses - Assessed for skeletal development and abnormalities.

Statistics:

The following data types were analyzed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

Indices:

Litter size and survival indices

Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100
Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100
Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values.

Historical control data:

Fetal examinations - See tables 1-3

Species/strain/source - Rat/ Crl:CD(SD)/Charles River (UK)
Necropsy date range - Aug 2017-Apr 2018
Number of studies - 6
Study types - Main 6

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs seen in association with dosing females with Oxirane, mono[(C12-14-alkyloxy)methyl] derivs at 0, 100, 330 or 1000 mg/kg/day. There were no clinical signs considered related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall bodyweight gain (GD 6-20) was slightly low (90% of controls) in females treated at 1000 mg/kg/day.
Group mean bodyweight stasis was observed for animals treated at 1000 mg/kg/day after the first administration; however, bodyweight gains were generally similar to Controls thereafter.
Body weight and body weight gain of females treated at 100 or 330 mg/kg/day and gravid uterine weight at all dose levels were unaffected.
As body weight changes (GD 6-20) were slightly low at 1000 mg/kg/day and gravid uterine weights were unaffected, the resulting adjusted body weight gains (6-20) were slightly low.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES:
1) BODY WEIGHT - GROUP MEAN VALUES DURING GESTATION
2) BODYWEIGHT CHNAGE - GROUP MEAN VALUES DURING GESTATION
3) GRAVID UTERINE WEIGHT, ADJUSTED BODYWEIGHT AND ADJUSTED BODYWEIGHT CHANGE - GROUP MEAN VALUES ON DAY 20 OF GESTATION

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment at all treated groups.

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 20 after mating, macroscopic examination revealed findings associated with the thickening of the forestomach in 15 females; in 3 females depressions in the forestomach were also apparent for females treated at 1000 mg/kg/day.
There were no other macroscopic findings, considered to be related with treatment, in adults.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

Rats do not abort

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on pre/post-implantation loss, number of live young or sex ratio.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE: "LITTER DATA - GROUP MEAN VALUES ON DAY 20 OF GESTATION"

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Dead fetuses:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE: "LITTER DATA - GROUP MEAN VALUES ON DAY 20 OF GESTATION"

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At macroscopic examination, one female treated at 1000 mg/kg/day (4F 80) was found not to be pregnant; therefore, reproductive assessment was based on 20, 20, 20 and 19 animals at 0,
100, 330 or 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

necropsy findings

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: Forestomach

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on placental, litter or fetal weights.

PLEASE REFER TO THE ATTACHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES ON DAY 20 OF GESTATION"

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE: "LITTER DATA - GROUP MEAN VALUES ON DAY 20 OF GESTATION"

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE: "LITTER DATA - GROUP MEAN VALUES ON DAY 20 OF GESTATION"

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

PLEASE REFER TO THE ATTACHED TABLES:
1) FETAL EXAMINATIONS - MAJOR ABNORMALITY FINDINGS - GROUP INDICES
2) FETAL EXAMINATIONS_MINOR SKELETAL AND VARIANT FINDINGS- GROUP INDICES

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

PLEASE REFER TO THE ATTACHED TABLE: "FETAL EXAMINATIONS_MINOR VISCERL ABNORMALITY AND NECROPSY FINDINGS-GROUP INDICES

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There was no effect of treatment on pre/post-implantation loss, number of live young or sex ratio.
There was no effect of treatment on placental, litter or fetal weights.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: sternebrae, Dorsal fat pad haemorrhage, Cerebellum subdural haemorrhage,Thymus gland partially undescended right lobe, median liver lobe haemorrhage, Testis right malpositioned,

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

Key result

Developmental effects observed:

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

Conclusions:

In this study Oxirane, mono[(C12-14-alkyloxy)methyl] derivs was well tolerated and the maternal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day. There was no adverse effect on embryo-fetal survival, development or growth, therefore the fetal no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was an assessment of the influence of Oxirane, mono[(C12-14- alkyloxy)methyl] derivs, an industrial chemical, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat.

Three groups of 20 females received Oxirane, mono[(C12-14-alkyloxy)methyl] derivs at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

There were no clinical signs that were considered related to treatment and no signs seen in association with dosing.

Overall body weight gains were slightly reduced at 1000 mg/kg/day. At 1000 mg/kg/day, there was group mean body weight stasis following initial treatment (Days 6 to 7 of gestation), thereafter subsequent body weight gains were largely unaffected.

There was no clear effect on food consumption.

There was no effect of treatment on gravid uterine weight and fetuses were macroscopically normal. At 1000 mg/kg/day, thickening of the forestomach was apparent in 15 females, with depressions on the forestomach in three of these females.

The number of corpora lutea, implantations, pre/post implantation loss, sex ratio of males to females and placental and fetal weights were unaffected by treatment.

Macroscopic examination of adult females on day 20 of gestation revealed findings associated with the thickening of the forestomach several females treated at 1000 mg/kg/day.

The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

Conclusion

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 375 mg/kg bw/day
Study duration:: subchronic
Species:: rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 200 mg/kg bw/day
Study duration:: subchronic
Species:: rat

Additional information

Justification for selection of Effect on developmental toxicity: via dermal route:
only existing study

Toxicity to reproduction: other studies

Additional information

In a 13-week dermal study in Fisher rats, gross and histopathologic examinations revealed no adverse effects on the ovaries and testes of animals dosed with the substance at 100 mg/kg/day.

The maternal and developmental toxicity ofOxirane, 2-((C12-14-alkyloxy)methyl)derivswas investigated by daily dermal administration in rats according to a protocol similar to the OECD guideline 414 and in compliance with GLP (Stump, 1997). During the study,Oxirane, 2-((C12-14-alkyloxy)methyl)derivswas applied at doses of1, 10, 50, 100 and 200 mg/kg/dto the clipped intact dorsal skin of each test animal for six hours per day, from gestation days 6 through 15.

All maternal animals survived to the scheduled necropsy on gestation day 20. The only treatment-related clinical sign observed was vocalization at the time of dosing in the 50, 100 and 200 mg/kg/day groups between gestation days 7 and 15. Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 1, 10, 50, 100 and 200 mg/kg/day groups were similar to the control group values. Food consumption, assessed as g/animal/day and g/kg/day, in the treated groups was unaffected by test article administration. At the scheduled necropsy, no test article-related internal findings were observed at any dose level.

Findings at the application site were limited to scabbing in two and six animals in the 100 and 200 mg/kg/day groups, respectively, and thickening in two of these high dose group animals. Intrauterine growth and survival were unaffected by test article administration at any dose level. Parameters evaluated included postimplantation loss, mean fetal body weight, viable litter size, fetal sex ratios and the mean numbers of corpora. lutea and implantation sites. Fetuses (litters) available for external morphological evaluation numbered 99(7), 110(8), 91(7), 79(7), 95(7) and 87(6) in the control, 1, 10, 50, 100 and 200 mg/kg/day groups, respectively. One control group fetus had macroglossia. No other external malformations and no external developmental variations were observed in fetuses in this study.

In conclusion, localized dermal irritation was observed in a dose-related manner at dose levels of 50, 100 and 200 mg/kg/day. No maternal toxicity or developmental toxicity was apparent at any dose level. Based on the results of this study, the no-effect level for dermal irritation was considered to be 10 mg/kg/day and the no-effect levels for maternal toxicity and developmental toxicity was considered to be 200 mg/kg/day.

Justification for classification or non-classification

Based on the above stated assessment, the substance does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and accordingCLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Oxirane, mono[(C12-14-alkyloxy)methyl] derivs.

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Formulation Analysis

SUMMARY

Reference 3

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Toxicity to reproduction: other studies

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all