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Toxicological information

Developmental toxicity / teratogenicity

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzophenone
- Identity: confirmed by infrared (IR) spectroscopy and mass spectrometry.
- Physical state: white solid, crystalines
- Analytical purity: >99% (Within 30-days prior to the initiation of this study, a bulk chemical sample was reanalyzed by liquid chromatography. The bulk chemical purity was 97.7% relative to a frozen reference sample)
- Molecular Formula: C13H100
- Molecular Weight: 182.22
-Lot/Batch No.: 10803KG
-Storage: Ambient temperature (-23-28°C); sealed and protected from light

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
-Age at receipt: Females 8-10 weeks Males: 10-12 weeks
- Weight: Maternal body weights, for confirmed pregnant females used in this study, ranged from 3.124-4.473 kg on gd 0
- Fasting period before study: no
- Housing: individually housed in in stainless steel cages with mesh flooring
- Diet: Feed was rationed at ~65 g during the first 24 hours after arrival (gd 1 and 2). For the following 24 hours, females on gd 2 received ~ 125 g of feed, and feed was available ad libitum for all females from gd 3 to scheduled sacrifice.
- Water: ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
The total range for temperature was 65.3-70.6 OF during Replicate I and 65.3-72.1 OF for Replicate II.
The total range for relative humidity was 45.3-64.6% for Replicate I and 36.7-59.9% for Replicate II.
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: Gestational day 0 dates for the first of two study replicates were April 18-
19, 1999. Evaluation of fetal heads and skeletons was completed on July 26, 1999.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Benzophenone was formulated in aqueous 0.5% methyIcellulose. Dose formulations were stored in sealed, amber glass bottles at room temperature, and mixed well prior to dosing. Formulations were administered within the 30-day period of proven stability.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Suspendability studies indicated that benzophenone was suspendable in 0.5% methylcellulose in water at a level of 200 mg/mL.
- Concentration in vehicle: 0, 1.67, 8.33 and 15.0 mg/mL
- Amount of vehicle (if gavage): 3 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In connection with a previous developmental toxicity screening study in rats (NTP, 1999), the homogeneity of benzophenone in 0.5% methyIcellulose was evaluated at a concentration of 60 mg/mL. Samples from the top, middle and bottom were analyzed by high performance liquid chromatography (HPLC).The assay values were 114%, 102% and 104%, respectively. Thus, test chemical concentration and homogeneity for this chemical/vehicle mixture were verified. During the study, four sets of dose formulations were prepared (two sets per study replicate), and aliquots were submitted for verification of concentration before each period of use. In addition, the first and third mixes (Rep I, Mix 1 and Rep II, Mix 1) were analyzed after the period of use.
Details on mating procedure:
- Impregnation procedure: Females were naturally-mated by the vendor prior to shipment to RTI. For each replicate, animals arrived at RTI on gd 1 (24 females) or gd 2 (24 females).
- M/F ratio per cage: 1.1
- Proof of pregnancy: yes, successful cohabitation, referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
Day 6 trough 29 of pregnancy
Frequency of treatment:
Once daily
Duration of test:
On gd 30, timed-mated females were sacrificed
Doses / concentrations
Remarks:
0, 5, 25 or 45 mg/kg bw
No. of animals per sex per dose:
Separate shipments of 48 animals were used for each replicate of the study. Females were naturally-mated by the vendor prior to shipment to RTI. For each replicate, animals arrived at RTI on gd 1 (24 females) or gd 2 (24 females). A total of 24 naturally-mated females per group (12 per replicate per group) were assigned to this study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection was based on a screening study in which NZW rabbits were treated by gavage with 0, 25, 50, 75, 100, or 125 mg benzophenone/kg of body weight/day on gd 6 through 29 (NTP, 2004). In the screening study, evidence of maternal toxicity was found at all
doses, and the upper three treatment groups were terminated early due to excessive maternal toxicity or early delivery of litters. There was no statistically significant evidence of developmental toxicity at doses up to 50 mg/kg/day (NTP, 2004).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Females were observed for clinical condition at least once/day from gd I or 2 (day of animal arrival) through gd 5 (prior to initiation of dosing). From gd 6 through 29, females were observed for clinical condition and signs of toxicity at dosing and approximately 1-2 hours after each daily dose. On gd 30, females were observed for clinical condition at weighing and at scheduled termination.

BODY WEIGHT: Yes
- Time schedule for examinations: on the mornings of gd 0 (vendor), 3, 6 through 30, and immediately following sacrifice on gd 30.

FOOD CONSUMPTION: Yes, on gd 3, 6, 9, 12, 15, 18, 21, 24, 27, 29, and 30.

WATER CONSUMPTION No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: yes

OTHER: The body, liver, paired kidneys, and gravid uterus of each timed-mated female were weighed. Thoracic and abdominal cavities were examined. Sections of all maternal livers, maternal kidneys, as well as any maternal organs that displayed gross pathology, were saved in 10% neutral buffered formalin for optional histopathology
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: Dead fetuses, and live fetuses. Dead fetuses were counted, weighed, and discarded. Uteri which presented no visible implantation sites were stained with ammonium sulfide (10%) in order to visualize any implantation sites which might have undergone very early resorption.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: All fetal carcasses were sexed and examined for visceral morphological abnormalities using a fresh tissue dissection method
- Head examinations: Heads from approximately one-half (50%) of the fetal carcasses were fixed and decalcified in Bouin' s solution and subsequently examined using a free-hand sectioning technique (Wilson, 1965).
- Skeletal examinations: All fetal carcasses were eviscerated, and the skeletons macerated and stained with alcian blue/alizarin red S stain (Marr et ai., 1988). Fetal skeletons were examined for morphological abnormalities.
Statistics:
Parametric statistical procedures were applied to quantitative continuous data (e.g., maternal body weights, fetal body weights, feed consumption, etc.). Bartlett's test for homogeneity of variance was reported using an alpha level of p=O.OOl. General Linear Models were applied to the Analyses of Variance (ANOVA) and the Tests for Linear Trend. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967). For litterderived percentage data, the ANOV A was weighted according to litter size. When a significant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test (Dunnett, 1955; 1964) was used to compare each treatment group to the control group for that measure. A one-tailed test (i.e., Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a twotailed test was used for maternal body and organ weight parameters, maternal feed consumption, fetal body weight, and percent males per litter. Data for any measure which showed a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOV A were presented as Mean ± SEM for each cell in the ANOV A design. Dose effects within each replicate were further evaluated using a one-way ANOVA and Test for Linear Trend. Dunnett's Test was applied for pairwise comparisons if the one-way ANOVA was significant (<0.05). Nominal scale measures were analyzed by ChiSquare Test .When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between each treatment group and the control group. The alpha level for each statistical comparison (other than Bartlett's test above) was 0.05. The significance level for each trend test or pairwise comparison (one- or two-tailed) was reported if it reached p<0.05 or p<0.01.
Indices:
not applicable
Historical control data:
Present for comparison, if indicated

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Dose-related incidence of maternal mortality and early termination of pregnancy (i.e., abortion or early delivery), as well as reduced body weight, weight gain and feed consumption during late gestation.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no maternal toxicity was observed
Key result
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
number of abortions

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Average fetal body weight per litter exhibited decreasing dose-response trends (males, females and both sexes combined). At the high dose, significant reductions were noted for male fetal body weight (81 % of control weight), female fetal body weight (85% of control weight) and both sexes combined (83% of control weight). There was an apparent decrease in the number of fetuses with extra rib(s) on Lumbar I with increasing doses of benzophenone. However, the apparent decrease was due to the smaller number of surviving litters at the mid and high-dose groups. Thus, there were no statistically significant differences among groups when the incidence of this finding was expressed as a percentage of rabbit fetuses (or percentage of litters) in which this finding occurred.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For further details see attached background material

Applicant's summary and conclusion

Conclusions:
The maternal toxicity LOAEL was 25 mg/kg bw and the NOAEL was 5 mg/kg bw. The developmental toxicity LOAEL was 45 mg/kg bw (based on reduced fetal body weight) and the NOAEL was 25 mg/kg bw.
Executive summary:

A developmental toxicity study in rabbits was performed according to US EPA Guideline requirements, which widely complies with OECD 414. Pregnant rabbits were treated from day 6 through 29 of pregnancy at daily dosages of 0, 5, 25 or 45 mg/kg bw. Maternal body weight and feed consumption showed decreasing trends. There were no changes in the weights of the gravid uterus, liver, or kidney of treated does. Benzophenone had no significant adverse effect on prenatal viability in litters that were carried until scheduled termination on gestational day 30. The ability of does to successfully carry their pregnancies was clearly compromised in a dose related manner (0 mg/kg, 24/24; 5 mg/kg, 24/24; 25 mg/kg, 19/22; 45 mg/kg, 12/19). Fetal body weight was significantly decreased in the 45 mg/kg group only. Developmental toxicity was noted only in the presence of well-defined maternal toxicity. There was no evidence for selective susceptibility of the conceptus relative to the pregnant dam.