Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1991/05/07-1991/06/12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 414.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene
EC Number:
926-273-4
IUPAC Name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene

Test animals

Species:
rat
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Sex: Male (50), Female (100)
Weight (Female): 242 - 340 g (day 0)
Age at study initiation: Approximately 10-11 weeks
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 Chow, ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 21d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Mated females were dosed once daily by oral gavage from GD 6 through GD 15. Dose levels were based on the most recent body weights. Doses were administered at 0, 75, 150, and 450 mg/kg and dose volumes were 5 ml/kg for all groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
gas chromatography verification
Details on mating procedure:
Females were placed in males’ cages in a 1:1 ratio based on sequential ID numbers. Mating was confirmed the following morning by observation of a copulatory plug or by the presence of sperm in a vaginal rinse. The day mating was confirmed was the female’s day 0 of gestation (GD 0). After confirmed mating, the female was returned to her own cage and a new female was placed into the male’s cage until the required number of mated females was obtained by continuous cohabitation.
Duration of treatment / exposure:
Mated females were dosed once daily by oral gavage from GD 6 through GD 15. Dose levels were based on the most recent body weights. Doses were administered at 0, 75, 150, and 450 mg/kg and dose volumes were 5 ml/kg for all groups.
Frequency of treatment:
Mated females were dosed once daily by oral gavage from GD 6 through GD 15.
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 150, and 450 mg/kg
Basis:
other: oral gavage
No. of animals per sex per dose:
Male (50), Female (100) total; 25 Females per dose
Control animals:
yes
Details on study design:
PARAMETERS Analyzed for Statistical Differences
- mean body weight and gravid uterine weight of pregnant animals
- mean body weight change of pregnant animals
- mean food consumption of pregnant animals
- mean number of corpora lutea per dam
- mean number of implantations per dam
- mean number of resorptions per dam
- mean number of live fetuses per litter
- mean number of live male fetuses per litter
- mean number of live female fetuses per litter
- mean number of dead fetuses per litter
- mean number of malformed fetuses per litter
- mean number of affected (resorbed, dead or malformed) offspring per litter
- mean number of fetuses with developmental variations per litter

Live fetuses as fraction of implants, dead fetuses as fraction of implants, and resorptions as fraction of implants, were calculated for each litter. Fractions were transformed by Cochran’s transformation followed by the arc sine transformation. The raw fractions and the transformed fractions were tested for statistical significance. Transformed percentages (with statistical analyses) were not presented in the report since statistical significance was not found.

Examinations

Maternal examinations:
All females were examined by a gross necropsy.
Ovaries and uterine content:
Uterine weight with ovaries attached was measured at necropsy. Uterine contents were examined and the numbers and locations of implantation sites, early and late resorptions, live, dead, and externally malformed fetuses were recorded. Corpora lutea were counted and recorded. The uteri of all apparently non-pregnant dams were stained to confirm pregnancy status.
Fetal examinations:
Each live fetus was weighed and examined externally for gross malformations, including cleft palate. All live fetuses were euthanized by intramuscular injection into the tongue with sodium pentobarbital prior to internal examination. The sex of each live fetus was determined by external examination and confirmed internally only on those fetuses receiving visceral examinations.

Approximately one-half of the fetuses of each litter were decapitated. These heads were preserved in Bouin’s solution for at least two weeks. Free-hand razor blade sections of the heads were examined for the presence of abnormalities. The viscera of all decapitated fetuses were also examined by fresh dissection. The remaining fetuses were eviscerated, processed for skeletal staining with Alizarin red, and examined for the presence of malformations and ossification variations. Representative malformations were photographed.
Statistics:
STATISTICAL METHODS (see "Further details on study design" field for parameters measured)

Bartlett’s test of homogeneity of variance was used to determine if the groups had equivalent variances at the 1% level of significance.

1. If the variances were equivalent, the hypothesis that there was no difference in response between the groups was tested using a one-way ANOVA, and if it was significant, Dunnett’s test was performed to determine which treatment groups differed from the control. A linear regression to test for a dose response was also performed and tested for lack of fit to the regression.

2. If the variances were not equivalent, then a Kruskal-Wallis (non-parametric) test was performed to determine if the treatment effects were equivalent. If there was a difference, a rank sum comparison was used to determine which treatment groups differed from the control. Jonckheere’s test for ordered response was also performed.


The following were calculated and analyzed for statistical significance:
- incidences of individual and total malformations
- incidences of individual and total variations
A standard chi square analysis was performed to determine if the proportion of incidences differed between the groups tested. Each group was then compared to each control group using a 2 x 2 Fisher Exact test. Armitage’s test for linear trend in the dosage groups was performed.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL IN-LIFE OBSERVATIONS
Low incidences of alopecia, urine staining, nasal discharge, red material around snout, dry rales, swollen area, soft stool, and mal-occluded or broken incisors were observed intermittently throughout the groups and considered incidental and unrelated to treatment. All animals survived to the scheduled cesarean sections on 21G. One animal in each of the low and high dose groups, and three animals in the mid-dose group were not pregnant.

BODY WEIGHTS
There were no significant differences in mean body weight at any interval between treated groups and controls. However, there was a noted body weight gain suppression observed in the treated groups when compared to controls, with the high dose group demonstrating a statistically significant change (GD 6-15). Overall body weight differences were not significant (GD 0-21).

FOOD CONSUMPTION
A significant decrease in mean maternal food consumption was observed in the high dose group at the GD 6-9 interval when compared with controls. Overall (GD 0-21) there were no significant changes observed in any of the treated groups when compared to the control group.

ORGAN WEIGHT
No significant changes.

MATERNAL NECROPSY OBSERVATIONS
The incidental findings observed at necropsy were not attributed to the treatment.

UTERINE IMPLANTATION DATA
There were no statistical differences between treated and control animals for any uterine implantation parameter.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 450 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHTS
There were no statistical differences between treated and control groups.

FETAL VARIATIONS AND MALFORMATIONS
There were no statistical differences in the incidences of fetal variations or malformations were observed in treated groups when compared with controls on either a per fetus or per litter basis. Visceral variations were observed in all groups included dilated renal pelvis and distended and/or convoluted ureters. Umbilical artery and/or vein on the left side of the urinary bladder were observed in one fetus in the control group and in two fetuses from two litters in the mid-dose group. No innominate artery was observed in two fetuses from one litter in the low dose group. Skeletal variations were observed throughout the groups and consisted primarily of hypoplastic or misshapen elements of the skull, sternebrae, vertebrae, or ribs. These findings are common in gestation day 21 rat fetuses and the incidence or severities were not considered treatment-related.

The majority of malformations were observed in a single instance in the low and high dose as well as additional malformations in control animals. External malformations included cleft palate, low set ears, anury, and anal atresia. Visceral malformations were also correlative to those fetuses with external malformations. Skeletal malformations were observed throughout the dose groups and consisted primarily of single instances of malformed or fused skull bones, agenesis of the ribs, vertebral arches or centra, and duplicates or extra bones of the hind paw. All malformations were considered spurious and unrelated to treatment.

FETAL SKELETAL OSSIFICATION SITES
There was a statistically significant decrease in the mean number of ossification sites in the proximal phalanges of the hindpaw in the high dose group. However, the number falls within the normal range of 0-3 and the decrease in the high dose group was not considered biologically significant.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The maternal NOAEL was determined to be 150 mg/kg and a developmental NOAEL was determined to be greater than 450 mg/kg. MRD-90-884 was not teratogenic under the conditions of this study and would not warrant classification under either the EU GHS or the EU Requirements for dangerous substances and preparations guidelines.
Executive summary:

The objective of this study was to determine the teratogenic potential of MRD-90-884 when administered to pregnant rats during gestational day (GD) 6 through GD 15.  The test material was administered by oral gavage at doses of 75, 150, and 450 mg/kg at a volume of 5 ml/kg. There were no treatment-related clinical in-life observations or any observable abnormalities in the dams for any dose group.  All animals survived to scheduled cesarean sections on GD 21.   There were no significant differences between treated and control animals for any uterine implantation parameter.  Mean fetal body weights for treated groups were comparable to control groups.  There were no statistically significant differences in the incidences of total or individual variations or malformations when analyzed on either a fetus or litter basis.  The maternal NOAEL was established as 150 mg/kg and a developmental NOAEL was established as greater than 450 mg/kg.  MRD-90-884 was not teratogenic under the conditions of this study and would not warrant classification under either the EU GHS or the EU requirements for dangerous substances and preparations guidelines.