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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

Alkyl (C12-C14) glycidyl ether
Label: Epoxide 8
51% C12 Glycidyl ether
21% C14 Glycidyl ether
5% C16 Glycidyl ether
13% N +1's
4% alpha addition Products
94% total active (label claim)
- Physical state: Clear colorless liquid
- Storage condition of test material: ambient

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)BR

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.

Administration / exposure

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.

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:

8

Details on study design:

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

Examinations

Maternal 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 fmdings.

Fetal examinations:

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:

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

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.