Diethylbenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This is a GLP guideline study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA Guidelines (Guidelines for registering chemicals in the U.S. , Health Effects Guidelines, Toxic Substances Control Act (TSCA) issued August 1982)

Deviations:

no

Remarks:

Not specified in report

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diethylbenzene

EC Number:

246-874-9

EC Name:

Diethylbenzene

Cas Number:

25340-17-4

Molecular formula:

C10H14

IUPAC Name:

diethylbenzene

Details on test material:

IUCLID4 Test substance: as prescribed by 1.1 - 1.4

MCS 2313; Mixed Diethylbenzenes (CAS No. 25340-17-4)
Reference Number: Lot # NBP 3529297
Source: Monsanto Company
Appearance: Clear, colorless liquid
Isomeric distribution and Purity: Not stated.

Control (vehicle) Article: Mazola Corn Oil
Reference No.: Lot#JUL1592A

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Animal Husbandry
Animal housing and care were based on the standards established by the American Association for Accreditation of Laboratory Animal Care (AAALAC) and the guidelines set forth in the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23, 1985.

1. Animal Receipt, Identification and Housing
One hundred and thirty Sprague-Dawley Crl:CD@BR VAF/PIusa rats were received at SLS on October 10, 1991, from Charles River Laboratories, Inc., Portage, MI, for this study. At the time of receipt, each rat was identified with a metal ear tag displaying an unique number. Animals were housed individually during acclimation and while on study (except during cohabitation) in suspended stainless steel cages.

2. Acclimation
Animals were examined upon receipt and daily thereafter during acclimation for signs of physical or behavioral abnormalities. Only healthy animals were maintained for possible assignment to the study. Mortality checks were performed twice daily, in the morning and afternoon. Individual body weights were determined on the day following receipt and just prior to cohabitation for mating. Animals were acclimated to the laboratory conditions for a period of 12 days prior to cohabitation.

3. Diet and Drinking Water
Purina Certified Rodent Meal@ #5002 and municipal tap water were provided to each animal ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants. The lot number and expiration date of each batch of food used during the study were recorded. The tap water was purified by reverse osmosis or deionization (backup purification system) and supplied to the animals by an automatic watering system. Water supplying the facility is analyzed on an annual basis for contaminants according to SLS Standard Operating Procedures. The results of the feed and water analyses are maintained at SLS.

4. Environmental Conditions
Animals were housed throughout the study in an environment-controlled room with a 12-hour light/12-hour dark cycle. The controls were set to maintain a room temperature of 64-7g0 F and a relative humidity of 40-70%. The room temperature and relative humidity were determined and recorded a minimum of once daily. On nine days, the relative humidity was outside the specified range by -11 to +2%. These occurrences were not considered to have had an impact on the outcome of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Preparation of Dosing Mixtures
A specified amount of the test article for each dose group was weighed into a volumetric flask. A sufficient quantity of corn oil was added to each flask to achieve the desired concentrations. Flasks were inverted approximately 20 times prior to adding a stir bar. Mixtures were stirred for 10 minutes and dispensed into daily aliquots. The mixtures were prepared weekly and stored at room temperature. Daily aliquots were stirred for 10 minutes prior to dispensation.

All doses were given at a constant volume of 5 ml/kg.

Dosing preparations were administered orally, by gavage, as a single dose daily from gestation day 6 through gestation day 15. Individual doses were adjusted based on the most recent body weight data. Oral administration of the test article was selected since this is a potential route of human exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability, Homogeneity and Concentration Analyses
Prior to study initiation, a modified version of the analytical method provided by the Sponsor was validated at SLS. Prestudy homogeneity and stability analyses were performed on concentrations of the test article which encompassed those used in this study. In addition, each fresh preparation of the dosing mixtures was analyzed for verification of test article concentration.

Experimental Svstem
1. GC System
Gas Chromatograph: Perkin-Elmer Sigma 2
Column: 6 feet x 2 mm ID, glass, packed with 5% SP-2100 on 100/120 mesh supelcoport
Detector: Flame Ionization Detector (FID)
Detector Temperature 300°C
Injector Port Temperature: 250°C
Column Temperature: 750C for 5 minutes then raise to 225C at 10°C/min
Carrier Gas: Helium at 40 mL/minute
Flame Gas: Hydrogen at 30 mL/minute; Air at 330 mL/minute
Data System: Hewlett Packard Model 33968
Injection Volume: 2 uL

Solutions and Reagents
Internal Standard Solution: Prepared by dissolving approximately 0.125 g of Tetradecane in a 50 mL volumetric flask with acetone.

Stock MCS 2313 Standard Solution: Prepared by dissolving approximately 0.025 g of bulk MCS 2313 in a 25 mL volumetric flask with acetone.

MCS 2313 Standard Curve Solutions: Prepared by diluting separate volumes of stock MCS 2313 standard solution with 1 mL of internal standard solution and acetone to a total volume of 10 mL. The final concentrations of MCS 2313 were approximately in the range of 0.1-0.4 mg/mL.

Analytical Dosing Solutions: Prepared by diluting dosing solutions with 1 mL of internal standard solution and acetone to a total volume of 10 mL. The final concentration of MCS 2313 was 0.2 mg/mL.

Analytical Procedures
In order to obtain accurate results, both sample and standard curve solutions were made to contain exactly the same amount of internal standard prior to analysis.
1. Standard Curve
The response factor of each standard curve solution was determined and plotted as a function of concentration to generate a standard curve.

2. Sample Analvsis
In order to best utilize the standard curve, the samples were diluted to a concentration in the range of the standard curve. This dilution was performed in duplicate to ensure accuracy of the analytical method. The response factor of each sample was measured and concentration was determined by linear fit to the standard curve. The actual concentration of the sample was further determined by multiplying by the dilution factor.

Homogeneity Analysis
The homogeneity of 1 and 200 mg/mL solutions was determined by analyzing the aliquot taken from the upper, middle and lower portions of the sample reservoir. The sample solution was determined to be homogeneous if the mean value of the actual concentrations of each sample taken from each region of the reservoir was within +/- 10% of the nominal concentrations.

Stability Analysis
The stability of 1 and 200 mg/mL solutions was determined by analyzing the samples on 3 and 8 days post-preparation. The sample solution was
determined to be stable if the mean value of the actual concentrations of each sample was within +/- 10% of the nominal concentrations.

Concentration Verification Analvsis
The 0, 4, 20, and 40 mg/mL solutions used for dosing in the teratology study in rats were analyzed weekly during the study.

C. Calculations
Standard curve linearity (correlation coefficient) and concentration determinations were accomplished using a TI59 (Texas Instruments) linear regression program.

Details on mating procedure:

Mating and Group Assianment
At the conclusion of acclimation, the animals were weighed and examined. Females determined to be suitable test subjects, based on age, healthy appearance, and body weight, were cohabitated with resident Sprague-Dawley Crl:CDBR VAF/PIus male rats. At the initiation of breeding, all females were approximately 92 days of age with body weights ranging from 220 to 260 g. Evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was confirmed was designated as day 0 of gestation. At that time, the female rats were assigned consecutively, in a block design, to study groups.

Duration of treatment / exposure:

Day 6 through 15 of gestation

Frequency of treatment:

7 days/week

Duration of test:

Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 mated female rats/group

Control animals:

yes, concurrent vehicle

Details on study design:

This study was performed to detect and evaluate the potential embryotoxic, fetal toxic or teratogenic effects of MCS 2313 when administered orally to pregnant rats during the period of major organogenesis. The study design consisted of a vehicle control and three treatment groups. Each group contained twenty-five mated female Sprague-Dawley rats. The test article was mixed with corn oil and administered at dose leveis of 20, 100 and 200 mg/kg/day from gestation day 6 through gestation day 15. All doses were given at a constant volume of 5 ml/kg. Control animals were administered corn oil under the same experimental conditions and at an equivalent dose volume. The animals were observed daily for clinical signs of toxicity. Body weights and food consumption were measured on gestation days 0, 6, 9, 12, 16 and 20. Surviving females were sacrificed on gestation day 20 and subjected to cesarean section. Fetuses were individually weighed, sexed and examined for external, visceral and skeletal abnormalities.

Examinations

Maternal examinations:

Parameters Evaluated
1. Clinical Observations
During the experimental period, all animals were observed daily for clinical signs of toxicity including physical or behavioral abnormalities. Mortality checks were performed twice daily, in the morning and afternoon, In addition, during the treatment period, the rats were observed between one-half and two hours following dosing for detection of overt signs of toxicity.

2. Body Weights
Individual body weights were measured on gestation days 0, 6,9, 12, 16 and 20. Body weight changes were calculated for the following gestation intervals: 0-6, 6-9, 9-12, 12-16, 16-20, 6-16 and 0-20.

3. Food Consumption
Individual food consumption was measured during gestation days 0-6, 6-9, 9-12,12-16, 16-20,6-16 and 0-20. Food consumption was calculated and reported as grams/animal/day and grams/kg/day.

4. Unscheduled Death
Females that died during the study were necropsied. Body cavities (cranial, thoracic, abdominal and pelvic) were opened and examined and abnormalities were recorded. Uterine contents were examined and the number of implantation sites was recorded. The number of corpora lutea on each ovary was also recorded.

5. Scheduled Sacrifice and Cesarean Section
Surviving females were sacrificed on gestation day 20 by carbon dioxide inhalation and subjected to a gross necropsy examination. The thoracic, abdominal and pelvic cavities were opened and the viscera examined. Abnormalities were recorded and representative tissue samples (containing lesions) were preserved in 10% neutral buffered formalin for possible histological examination. The uterus was removed from the body, examined externally, weighed and then opened for internal examination. The number of viable and nonviable fetuses and early and late resorptions was then recorded beginning with the left distal uterine horn, noting the position of the cervix, and continuing up the right uterine horn. Corpora lutea were recorded for each ovary. Uteri with no macroscopic evidence of implantation were opened and placed in 10% aqueous ammonium sulfide solution for detection of early embryolethality as described by Salewski [I].

Ovaries and uterine content:

The uterus was removed from the body, examined externally, weighed and then opened for internal examination. The number of viable and nonviable fetuses and early and late resorptions was then recorded beginning with the left distal uterine horn, noting the position of the cervix, and continuing up the right uterine horn. Corpora lutea were recorded for each ovary. Uteri with no macroscopic evidence of implantation were opened and placed in 10% aqueous ammonium sulfide solution for detection of early embryolethality as described by Salewski [I].

Fetal examinations:

Fetal Morphological Examination
Fetuses were examined for external, internal (visceral), and skeletal abnormalities. Findings were classified based upon the severity of the anatomical
change(s) and their potential for interference with organ and/or body functions.

a. Fetal External Examination
Each fetus was examined for external abnormalities and the sex was determined, The fetuses were then weighed and tagged individually. The tags
displayed the study, dam and fetus numbers, as well as the fixative designation. The crown-rump length of each late resorption (evidence of autolysis) was measured and the tissue was then discarded.

b. Fetal Visceral Examination
Approximately one-half of the fetuses were fixed in Bouin's solution for subsequent visceral examination using Wilson's technique [2]. The examination was performed using a low power dissection scope.

c. Fetal Skeletal Examination
Approximately one-half of the fetuses were fixed in 95% isopropyl alcohol. Following fixation, the fetuses were macerated in 1-2% aqueous potassium
hydroxide solution, stained with Alizarin Red S, and cleared in glycerin [3]. The examination was performed using a low power microscope.

Statistics:

Continuous maternal and fetal data, including body  weights, body weight gain, food consumption, number of fetuses,  implantation sites and corpora lutea, were analyzed using one-way  analysis of variance (ANOVA) followed by Dunnett's test.  The  Mann-Whitney U test was used to compare post-implantation loss and  resorptions.  Fetal sex ratios were analyzed using the Chi-Square test.   Fisher's Exact test was used to analyze the incidence and number of fetal  malformations and variations utilizing the dam (litter) as the  experimental unit. All analyses were two-tailed with a minimum significance level of 5%.

Indices:

No data

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
No treatment-related mortality was observed during the study. One female in the 20 mg/kg/day group died shortly after dosing on gestation day 9. This death was attributed to an intubation error during dosing. All other females in the control and each MCS 2313 treatment group survived to scheduled sacrifice on gestation day 20. The pregnancy rate was 96% in the 20 mg/kg/day group and 100% in the control, 100 and 200 mg/kg/day groups.

Clinical Observations
No clinical signs of toxicity were observed during the study. Minor incidental findings, including reddish vaginal discharge, scabbing, urine staining, fecal staining, and hairloss, were observed at a low frequency throughout the control and treatment groups.

Body Weights and Body Weight Gains
Mean maternal body weight gain was statistically reduced at the 100 mg/kg/day level and statistically significant body weight loss occurred at the 200 mg/kg/day level during the first three days of dosing (gestation days 6-9). Subsequent weight gain at these levels was comparable to the control group. Mean weight gain at the 20 mg/kg/day level was similar to the control group throughout the study. The body weight loss at the 200 mg/kg/day level during gestation days 6-9 resulted in statistically reduced weight gain at this level for gestation days 6-16 and statistically decreased group mean body weights on gestation days 9, 12 and 16. Mean body weights were comparable among the control, 20 and 100 mg/kg/day groups throughout the study.

Net maternal body weight gain (day 0-20 body weight gain adjusted for gravid uterus weight) was also statistically reduced at the 200 mg/kg/day level when compared to the control group. Net weight gain was, however, comparable between the control, 20 and 100 mg/kg/day groups. No statistically significant differences were observed among the groups with respect to net maternal body weights (day 20 body weight minus gravid uterus weight).

Food Consumption
Food consumption calculated as grams/animal/day and grams/kg/day was statistically reduced in the 100 rng/kg/day group during gestation days 6-9 and in the 200 mg/kg/day group during gestation days 6-9, 9-12 and 6-16. At the 200 mg/kg/day level, food consumption was also reduced during days 0-20 when calculated as grams/animal/day. However, when calculated as grams/kg/day, food consumption was significantly increased during days 16-20 in the 200 mg/kg/day group. Food consumption was similar between the control and 20 mg/kg/day groups throughout the study.

Maternal Necropsv Observations
Necropsy findings for group 2 female #358, which died shortly after dosing on gestation day 9, consisted of a perforation through the glottis near the apex of the esophagus and approximately 5.0 ml of dark red fluid contents and associated blood clots in the thoracic cavity.

For females surviving to scheduled sacrifice, greenish-blue discoloration of the amniotic sac was observed in eleven 100 mg/kg/day females and all twenty-five 200 mg/kg/day females. No other significant changes were observed in surviving females at necropsy.

Cesarean Section Observations
Mean fetal body weight was statistically reduced at the 200 mg/kg/day level when compared to the control group. All other cesarean section parameters, including the mean number of corpora lutea, implantation sites, early resorptions, post-implantation toss, fetal sex ratios and mean gravid uterus weights, were comparable among the groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal data:  Mean fetal body weight was statistically reduced at the 200  mg/kg/day level when compared to the control group.  All other Cesarean  parameters were comparable among groups.  

No statistically significant or biologically meaningful differences were noted among the groups in the fetal examination data. Two 20 mg/kg/day fetuses and one fetus each from the 100 and 200 mg/kg/day groups were observed to have a single external or visceral abnormality which is known to occur spontaneously in this strain of rat. The incidence of developmental variations was generally comparable between the control and MCS 2313 treatment groups.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

2

Key result

Dose descriptor:

NOEL

Effect level:

> 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Range-Finding Results

This study was performed to provide information concerning the potential toxic effects of MCS 2313 when administered orally to pregnant rats for the purpose of selecting dose levels for a definitive teratology study: The study design consisted of a vehicle control and five treatment groups. Each group contained six mated female Sprague-Dawley rats. The test article was mixed with corn oil and administered at levels of 20, 50, 100, 150 and 200 mg/kg/day on gestation days 6 through 15. All doses were given at a constant volume of 5 ml/kg. Control animals were administered corn oil under the same experimental conditions and at an equivalent dose

- volume. The animals were observed daily for clinical signs of toxicity and weighed at specified intervals. Females were sacrificed on gestation day 20 and subjected to cesarean section. Fetuses were examined for external abnormalities, sexed, weighed individually and then discarded.

All females survived to scheduled sacrifice and no clinical signs of toxicity were observed. Mean maternal. body weight gain was slightly reduced at the 100, 150 and 200 mg/kg/day levels during the first: few days of dosing. Subsequent weight gain at these levels was similar to the control group. Mean weight gain at the 20 and 50 mg/kg/day levels was comparable to the control group throughout the study, At: necropsy, blue-green discoloration of the amniotic sac was observed in a dose-related manner at the 100, 150 and 200 mg/kg/day levels. All cesarean section parameters were comparable among the groups and no treatment-induced external malformationsor developmental variations were observed.

Therefore, based on the results of this study, dosage levels of 20, 100 and 200 mg/kg/day were selecLed for 'a definitive teratology study in rats with MCS 2313.

Analytical Chemistry Results

Prestudy analytical chemistry evaluations indicated that MCS 2313 was homogeneous and stable in corn oil for up to eight days when stored at room temperature. Analysis of dosing preparations resulted in average test article recoveries ranging from 99.3 to 103.2% indicating that the mixtures were accurately prepared.

Applicant's summary and conclusion

Conclusions:

Oral administration of MCS 2313 to pregnant rats during the major period of organogenesis produced dose-related maternal toxicity at dosage levels of 100 and 200 mg/kg/day and fetal toxicity at a dosage level of 200 mg/kg/day. MCS 2313 was not, however, teratogenic at any dosage level tested in this study. A dosage level of 20 mg/kg/day was considered a no-observed-effect level (NOEL) for maternal toxicity and a dosage level of 100 mg/kg/day was considered a NOEL for fetal toxicity.

Executive summary:

This study was performed to detect and evaluate the potential embryotoxic, fetal toxic or teratogenic effects of MCS 2313 when administered orally to pregnant rats

during the period of major organogenesis. The study design consisted of a vehicle control and three treatment groups. Each group contained twenty-five mated female

Sprague-Dawley rats. The test article was mixed with corn oil and administered at dose leveis of 20, 100 and 200 mg/kg/day from gestation day 6 through gestation

day 15. All doses were given at a constant volume of 5 ml/kg. Control animals were administered corn oil under the same experimental conditions and at an equivalent dose volume. The animals were observed daily for clinical signs of toxicity. Body weights and food consumption were measured on gestation days 0, 6, 9, 12, 16 and 20. Surviving females were sacrificed on gestation day 20 and subjected to cesarean section. Fetuses were individually weighed, sexed and examined for external, visceral and skeletal abnormalities.

No treatment-related mortality or clinical signs of toxicity were observed during the study. Mean maternal body weight gain and food consumption were statistically reduced at the 100 and 200 mg/kg/day levels during the first part of the treatment period. Weight gain and food consumption were comparable between the control and 20 mg/kg/day groups throughout the study. Greenish-blue discoloration of the amniotic sac was observed in a dose-related manner at the 100 and 200 mg/kg/day levels. Mean fetal body weight was statistically reduced at the 200 mg/kg/day level when compared to the control group. All other cesarean section parameters were comparable among the groups. No treatment-related malformations or developmental variations were observed in the study.

Oral administration of MCS 2313 produced dose-related maternal toxicity at dosage levels of 100 and 200 mg/kg/day and fetal toxicity at a dosage level of 200 mg/kg/day. MCS 2313 was not, however, teratogenic at any dosage level tested in this study. A dosage level of 20 mg/kg/day was considered a no observed-

effect level (NOEL) for maternal toxicity and a dosage level of 100 mg/kg/day was considered a NOEL for fetal toxicity.