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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Extracts (petroleum), light paraffinic distillate solvent
EC Number:
265-104-2
EC Name:
Extracts (petroleum), light paraffinic distillate solvent
Cas Number:
64742-05-8
IUPAC Name:
Extracts (petroleum), light paraffinic distillate solvent
Test material form:
liquid
Details on test material:
Extract, light paraffinic distillate solvent
CAS# 64742-05-8
Site# 7, Sample# 23
[WIL log no. 8470A]
Dark brown, viscous liquid
Specific details on test material used for the study:
Extract, light paraffinic distillate solvent
CAS# 64742-05-8
Site# 7, Sample# 23
[WIL log no. 8470A]
Dark brown, viscous liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 79 days (on receipt) plus 12 days acclimation
- Weight at study initiation: 222 - 301 g on gestation day 0
- Housing: Individually in stainless steel wire-mesh cages suspended above cage-board. Rats paired for mating in the home cage of the male with females returned to individual cages following positive evidence of mating.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum
- Water (e.g. ad libitum): Municipal supply ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71 +/- 5F (22 +/- 3 degrees C)
- Humidity (%): 50 +/- 20%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Sham, vehicle control and 4 test groups (5, 25, 150, 450 mg/kg/day)
Test substance formulations were prepared approximately weekly as single formualtions for each exposure level, divided into aliquots for daily dispensation and stored at room temperature protected from light. Test substance formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

On the day prior to the initiation of dosing administration, and as required thereafter, hair was clipped from the dorsal scapular area without abrading the skin. Repeat clippings were performed prior to or at least 2-4 hours after dose administration.
Different clippers were used for sham, vehicle control and test groups.
Test substance was applied evenly to ensure contact with ca. 10% of the body surface once daily for a period of 6 hours during gestation days 0-19.
Elizabethan style collars were applied to all animals to minimise ingestion of test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine homogeneity and resuspension homogeneity as well as 10-day room temperature stability at test substance concentrations of 1 and 500 mg/mL were previously conducted (Freer, 2011, WIL-402026) prior to the initiation of dosing and were not repeated during the study.
Details on mating procedure:
- Impregnation procedure: cohoused

- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: resident males were the same strain
- Proof of pregnancy: vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist; termed day 0 of gestation
Duration of treatment / exposure:
20 days
(days 0-19 of gestation)
Frequency of treatment:
Once daily
Duration of test:
20 days (duration of treatment)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
25 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
450 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
yes, sham-exposed

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for moribunidty and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at dose administration and 1-2 hours following sham or dose administration

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Individual food consumption was recorded on gestation days 0, 3, 6, 9, 12, 15, 18, and 20. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The cranial, thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined.
- The following tissues and organse were collected and placed in 10% neutral-buffered formalin: treated skin, brain, untreated skin (right hindlimb), thymus, liver (2 sections), all gross lesions
- Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfate solution for detection of early implantation loss.
- Liver brain and thymus were weighed from all animals
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: Placentae were also examined
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted.
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the vehicle control group (Group 2). In addition, the vehicle control group (Group 2) was compared separately to the sham control group (Group 1).
Indices:
Mean maternal body weights (absolute and net), body weight changes (absolute and net), food consumption, organ weights, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined)
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), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was evident at 25, 150, and 450 mg/kg/day as noted by clinical findings (yellow and/or red material on the urogenital area, red vaginal discharge, body pale and/or cool to the touch) at the daily examinations and/or 1 to 2 hours following dose administration and dermal observations of desquamation. One and 3 females in the 150 and 450 mg/kg/day groups, respectively, were euthanized in extremis between gestation days 14-18 as a result of lower mean food consumption with corresponding mean body weight losses and/or lower mean body weight gains noted in these groups generally throughout the treatment period; differences were generally statistically significant. Decreased mean food consumption with corresponding decrements in mean body weight gain were noted in the latter part of gestation in the 25 mg/kg/day group and correlated with the lower mean fetal weights noted in this group. Test substance-related macroscopic findings noted in the 150 and 450 mg/kg/day groups included dark red contents in the uterus, vagina, and/or cervix. Additionally, small thymus was noted at these exposure levels and corresponded to the statistically significantly lower mean thymus weights (absolute and relative to brain) noted in the 150 and 450 mg/kg/day groups. Statistically significantly lower mean thymus weights (absolute and relative to brain) were also noted in the 25 mg/kg/day group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Developmental toxicity was evident at 25, 150, and 450 mg/kg/day. Increased mean litter proportions of postimplantation loss (primarily early resorptions) with corresponding decreases in the mean numbers and litter proportions of viable fetuses were noted at 150 and 450 mg/kg/day. With the exception of 1 female that had 3 viable fetuses, all surviving females in the 450 mg/kg/day group had entirely resorbed litters (100% postimplantation loss with 0.0% viable fetuses). Therefore, comparative statistics were not performed on the mean fetal weights at 450 mg/kg/day. However, the fetal weights for the single litter in the 450 mg/kg/day group were lower than the vehicle control group. Additionally, mean male, female, and combined fetal weights in the 25 and 150 mg/kg/day groups were statistically significantly lower than the vehicle control group. These differences resulted in lower mean gravid uterine weights in the 25, 150, and 450 mg/kg/day groups; differences were statistically significant at 150 and
450 mg/kg/day. Test substance-related skeletal developmental variations (sternebra[e] nos. 5 and/or 6 unossified, reduced ossification of the skull, reduced ossification of the vertebral arches, and sternebra[e] nos. 1, 2, 3, and/or 4 unossified, and cervical centrum no.1 ossified) were noted in the 150 mg/kg/day group. The findings of sternebra(e) nos. 5 and/or 6 unossified, reduced ossification of the skull, and sternebra(e) nos. 1, 2, 3,
and/or 4 unossified were also noted for fetuses in the single surviving litter at 450 mg/kg/day. The aforementioned findings were considered indicators of developmental delay and correlated to the reduced fetal weights noted at 150 and 450 mg/kg/day.

Effect levels (fetuses)

Remarks on result:
other: see Details on results

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, an exposure level of 5 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity
and embryo/fetal development when extract, light paraffinic distillate solvent was administered by dermal application to bred Crl:CD(SD) rats.
Executive summary:

Maternal toxicity was evident in the 25, 150, and 450 mg/kg/day groups with adverse clinical and/or macroscopic findings and a higher incidence of dermal observations at these exposure levels. One and 3 females in the 150 and 450 mg/kg/day groups, respectively, were euthanized in extremis between gestation days 14-18 as a result of lower mean food consumption with corresponding mean body weight losses and/or lower mean body weight gains noted in these groups generally throughout the treatment period. Mean thymus weights (absolute and relative to brain) were noted in the 25, 150, and 450 mg/kg/day groups. No evidence of maternal toxicity was noted at 5 mg/kg/day.

Developmental effects were noted in the 150 and 450 mg/kg/day groups as evidenced by increased mean litter proportions of postimplantation loss (primarily early resorptions) with a corresponding decrease in the mean numbers and litter proportions of viable fetuses. In addition, lower mean male, female, and combined fetal weights were noted in the 25 and 150 mg/kg/day groups. Lower fetal weights were also noted for the single surviving litter in the 450 mg/kg/day group. Test substance-related fetal developmental variations (sternebra[e] nos. 5 and/or 6 unossified, reduced ossification of the skull, reduced ossification of the vertebral arches, sternebra[e] nos. 1, 2, 3, and/or 4 unossified, and cervical centrum no.1 ossified) were noted in the 150 mg/kg/day group and for surviving fetuses in the 450 mg/kg/day group and were indicators of developmental delay and correlated to lower fetal weights. Intrauterine growth and survival at 5 mg/kg/day and fetal morphology at 5 and 25 mg/kg/day were unaffected by test substance administration.