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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2002 to 29 January 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary guideline compliant study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
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)
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Remarks:
Compliance statement included
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate
EC Number:
219-207-4
EC Name:
7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate
Cas Number:
2386-87-0
Molecular formula:
C14H20O4
IUPAC Name:
7-oxabicyclo[4.1.0]hept-3-ylmethyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate
Specific details on test material used for the study:
- Name of test material (as cited in study report): cycloaliphatic epoxy resin ERL-4221
- Physical state: Clear, viscous, colourless liquid.
- Analytical purity: 92.3% - dose levels were adjusted to take acount of this purity
- Purity test date: 26 June 2000
- Lot/batch No.: 87068
- Expiration date of the lot/batch: Not documented
- Stability under test conditions: Stable under conditions outlined by the sponsor.
- Storage condition of test material: room temperature and protected from light.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: approximately 70 days old on arrival and circa 84 days old on study initiation
- Weight at study initiation: 223 - 307g
- Fasting period before study: Not applicable
- Housing: All rats were individually housed in clean, wire-mesh cages suspended above cage-board on arrival to the lab and until pairing. The rats were paired for mating in the home cage of the male. Following positive identification of mating, the females were returned to an individual suspended wire-mesh cage. Nesting material was not provided as animals were euthanized before parturition.
- Diet (e.g. ad libitum): Rodent LabDiet® 5002 provided ad libitum.
- Water (e.g. ad libitum): Reverse-osmosis purified (on-site) drinking water, delivered by an automatic watering system ad libitum.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2ºC to 22.4ºC
- Humidity (%): 34.3% to 48.1%
- Air changes (per hr): approximately 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark photoperiod

IN-LIFE DATES: From: 15 January 2002 To: 30 April 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola®
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the preparation of test article formulations, the appropriate amount of test article for each group was weighed into tared, calibrated storage containers for weight-to-volume (w/v) mixtures. The vehicle was added in small increments while stirring slowly (not creating a vortex). The containers were occasionally swirled by hand while adding the vehicle. A sufficient amount of vehicle was added to bring the volume to the calibration mark. The formulations were mixed slowly until uniform, using a magnetic stir bar. After uniformity was achieved, formulations were swirled prior to sampling and use for dose administration.

Fresh dosing formulations were prepared daily.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initial dose administration, samples (10 mL each) were collected from the top, middle and bottom strata of the 5, 25, 125 and 500 mg/kg/day dosing formulations and one sample (10 mL) was collected from the control group for homogeneity determination. Samples (10 mL each) for concentration analysis were collected weekly from each dosing formulation (including the control group). The test article concentration was measured using gas chromatography with a mass selective detector.
Homogeneity and stability of the test article formulations had been established in previous studies which are not summarised here.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Each female was placed in a suspended wire mesh cage with a resident male from the same strain and source.
- M/F ratio per cage: Study report indicates the ratio was one male and one female per cage.
- Length of cohabitation: Not documented

- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: copulatory plug and presence of sperm in vaginal smear indicated a positive mating which was referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Rats were treated from Day 6 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
20 days (from Day 0 of gestation).
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
25 female rats per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- Rationale for animal assignment (if not random): Not documented
- Other: On gestation day 20, all maternal rats were euthanized by carbon dioxide inhalation and a laparohysterectomy was performed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded from days 0 through 20 of gestation (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were 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, 12-20, 6-20 and 0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. On the occasions when food intake could not be measured for one of the days in a given interval (due to spillage, contamination with urine, weighing error, etc.), values were calculated using the appropriate number of days for that interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents were examined. The uterus and ovaries were excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all in utero fetuses, early and late resorptions and the total number of implantation sites were recorded. The placentae were also examined.

OTHER:
Organ Weights: The liver and kidneys were weighed.
Gravid Uterine Weight: Gravid uterine weight was collected and net body weight and net body weight change were calculated and presented for each gravid female.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The placentae were also examined.
Fetal examinations:
- External examinations: Yes: all per litter, including, eyes, palate and external orifices
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes: half per litter were placed in Bouin's fixative for subsequent soft tissue examination and the heads from the other half of the litter were examined by a mid-coronal slice.
Statistics:
Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, organ weights, numbers of corpora lutea, implantation sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant
(p<0.05) intergroup variance, Dunnett's test was used to compare the test article-treated groups to the control group.

Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution) were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test article-treated groups to the control group. The same statistical analysis was conducted to evaluate the significance of mean litter proportions of total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation.
Indices:
No information
Historical control data:
Historical Control Data was documented.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All animals survived to the scheduled necropsy on gestation day 20. A mean maternal body weight loss (2g) was observed in the 500 mg/kg/day group during gestation days 6-9 due to a mean body weight loss (11g) on the first day of dosing (gestation days 6-7). The difference from the control group gain was statistically significant (p<0.01). When the entire treatment period (gestation days 6-20) was evaluated, mean body weight gain in
this group (500mg/kg/day treatment group) was reduced (statistically significant at p<0.05) compared to the control group value which were considered to be test article related.
A mean body weight loss (6 g) was observed in the 125 mg/kg/day group on the first day of dose administration (gestation days 6-7). However, the loss was followed by a statistically significant (p<0.05) increase in mean body weight gain during gestation days 8-9 compared to that in the control group and the loss was not of sufficient magnitude to affect the mean body weight gain value during gestation days 6-9.Therefore, this transient loss, while attributed to the test article, was not considered to be adverse. No test article-related effects on mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights were observed in the 5 and 25 mg/kg/day groups.

Increased mean kidney weight in both the 500mg/kg/day and 125 mg/kg/day treatment groups.

In the 500mg/kg/day treatment group, lowere mean body weights and reduced body weight gain and food consumption was observed which at times achieved significance.

In the 125mg/kg/day treatment group, transient initial mean body weight losses and reduced food consumption were observed, some of which were statistically significant. However, due to the rapid amelioration of the effects, these observations were not considered to have an adverse effect.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In the 500 mg/kg/day treatment group, a statistically significant reduced mean foetal body weight was observed. In addition to this, increased skeletal developmental variations in the form of reduced mean litter proportion of cervical centrum no. 1 ossified, which was considered statistically significant. There was also increased mean litter proportions of unossified sternebrae, however, this was not considered statistically significant.
Intrauterine growth and survival in the 5, 25 and 125 mg/kg/day groups were unaffected by test article administration. Mean numbers of corpora lutea and implantation sites in all groups, including the control group, were similar.
One foetus in the 25 mg/kg/day and two foetuses in the 125 mg/kg/day groups had external malformations. The foetus in the 25 mg/kg/day group had bilateral anophthalmia, consisting skeletally of orbits that appeared smaller than normal. In the 125mg/kg/day treatment group, one foetus had anasarca while the 2nd foetus had a conjoined twin (two complete oedematous fetuses were joined along the ventral surface from the neck to the umbilicus). These malformations were considered spontaneous since no external malformations were observed in the 500 mg/kg/day group.
Soft tissue malformations were observed in two foetuses in the 125 mg/kg/day group. One foetus had a retroesophageal aortic arch in addition to an enlarged aorta. In the 2nd foetus, situs inversus was observed. These malformations were considered to be spontaneous since no soft tissue malformations were observed in the 500 mg/kg/day group. Several foetuses also displayed soft tissue findings that were not classified as malformations or developmental variations and were not included.
There were no skeletal malformations observed in foetuses in this study. However, in the 500mg/kg/day group, the mean litter proportion of cervical centrum no. 1 ossified (11.7%) in this group was reduced (statistically significant at p<0.01) compared to the control group value (25.7%). While this value was considered to be within the range of the historical control data, it indicated evidence of developmental delay in conjunction with reduced mean foetal body weights. The mean litter proportions for unossified sternebra(e) nos. 5 and/or 6 (26.4%) and unossified sternebra(e) nos. 1, 2, 3 and/or 4 (1.6%) were increased in the 500 mg/kg/day group compared to the control group values (7.6% and 0.3%, respectively). These values were also above the maximum values in the historical control data (21.4% and 1.0%, respectively). These skeletal variations were also attributed to the test article as further indication of developmental delay. Other skeletal developmental variations also occurred in all dose groups, however, no relationship to the test article was evident.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: reduced skeletal ossification

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

5, 25, 125 and 500 mg/kg bw/day doses were administered for 20 days from day 0 of gestation.  

At 500 mg/kg bw/day the maternal effects noted included - lower mean body weights (including a mean body weight loss from gestation days 6-7) and reduced body weight gain and food consumption; increased mean kidney weight. Necropsy revealed depressed renal cortex in one female at 125 and two females at 500 mg/kg bw/day and dilated renal pelvis in one control female. No treatment relationship was established. Maternal organ weights – mean kidney weights for 125 and 500 mg/kg bw/day groups were 9.6 and 12% higher than controls.

At 125 mg/kg bw/day the maternal effects observed included - Ttansient, initial mean body weight losses and reduced food consumption and increased mean kidney weight.

No test article-related effects were observed in the 5 and 25 mg/kg/day groups.

No maternal deaths occurred at any dose level. No adverse clinical signs of reaction to treatment at 5 to 500 mg/kg bw/day.

Test article-related developmental effects were noted in the 500 mg/kg/day group - Reduced mean foetal body weight and increased skeletal developmental variations (reduced mean litter proportion of cervical centrum no. 1 ossified and increased mean litter proportions of unossified sternebrae). Mean foetal weight at 500 mg/kg bw/day reduced below minimum for historical database.

No foetal effects were observed at the maternally toxic level of 125 mg/kg bw/day, or at lower doses. Intrauterine growth and survival was unaffected at 125 mg/kg bw/day.

No maternal effects noted at 5 or 25 mg/kg bw/day

No foetal effects evident at 5 or 25 mg/kg bw/day

No foetal malformations at any dose level 5-500 mg/kg bw/day

No developmental variations observed at 5, 25 or 125 mg/kg bw/day

Overall bodyweight gain in high dose dams was significantly reduced – an initial weight loss in GD 6-9 and then reduced weight gain GD 18-20. While it is unlikely the reduced gains on days 18-20 would have affected foetal development the initial weight loss in this group may have affected the intruterine foetal growth/development. No such effects were evident in the dams dosed at 125 mg/kg bw.

 

Skeletal developmental variations – in the 500 mg/kg bw group – limited to cervical centrum no 1 ossified, the mean litter proportion was reduced - 11.7% compared with 25% in controls. While these percentages were within the lab background range, it was considered the variation, in conjunction with reduced foetal bodyweight, was evidence of delayed development induced by the test article. The mean litter proportion for unossified sternebrae nos 5 and 6 (26.4%) was increased compared with controls (7.6%) and higher than the lab background upper range (21.4%) Similarly unossified sternebrae 1,2,3 and 4 incidence/proportion was increased (1.6%) compared with controls (0.3%) and historic background (1%). These sternebral changes were also considered indicative of delayed development at the high dose level.

  

It was concluded that the maternal NOAEL is 25 mg/kg based on increased kidney weight at 125 mg/kg bw/day.

The prenatal developmental NOAEL is 125 mg/kg based on observations of skeletal variations indicative of delayed development at 500 mg/kg bw/day only.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a dose level of 25mg/kg/day administered by oral gavage to rats was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dose level of 125mg/kg/day was considered the NOAEL for prenatal developmental toxicity.
Executive summary:

In the study conducted by Varsho (2007), the test article, Cycloaliphatic Epoxy Resin ERL-4221 was evaluated for its potential to act as a maternal and/or developmental toxicant. The test material was administered in the vehicle (corn oil) via oral gavage to four groups of 25 female Crl:CD®(SD)IGS BR rats. The rats were dosed once daily from gestation days 6 through 19 at concentration levels of 5, 25, 125 and 500 mg/kg/day. The control group received the vehicle on the same dosing regimen. All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on all females. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

All females survived to the scheduled necropsy. In the 500mg/kg/day group, maternal animals had lower mean body weights (including a mean body weight loss from gestation days 6-7) and reduced body weight gain and food consumption (occasionally statistically significant) and also had an increased mean kidney weight which was statistically significant. In the same dose group, a statistically significant reduced mean foetal body weight was also observed and increased skeletal developmental variations (reduced mean litter proportion of cervical centrum no. 1 ossified [statistically significant] and increased mean litter proportions of unossified sternebrae [not statistically significant]) were also observed.

In the 125mg/kg/day treatment group, a statistically significant increased mean kidney weight was observed in maternal animals. In addition to this, transient, initial mean body weight losses and reduced food consumption (some statistically significant). These observations were not considered to be adverse due to the rapid amelioration of the effects.

Based on the results of this study, a dose level of 25 mg/kg/day administered orally to rats was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dose level of 125 mg/kg/day was considered to be the NOAEL for prenatal developmental toxicity.