Reaction mass of divinylbenzene and ethylstyrene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Non-GLP study equivalent to OECD guideline 412.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of divinylbenzene and ethylstyrene

EC Number:

910-757-7

Cas Number:

N/A

Molecular formula:

Divinylbenzene: C10H10 Ethylstyrene: C10H12

IUPAC Name:

Reaction mass of divinylbenzene and ethylstyrene

Details on test material:

Divinylbenzene 55 (1 ot #840329-007) was obtained from The Dow Chemical Company production facilities, Midland, MI.

Capillary gas chromatographic analysis of the test material revealed that 53.72 of the material was divinylbenzene (15.4% para and 38.3% meta) and 42.9% was ethyl vinyl benzene (11.2% para and 31.7% meta).

Test animals

Species:

other: rat and mouse

Strain:

other: Fischer 344 rat and B6C3F1 mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Fischer 344 (F-344) rats and B6C3F1- mice (6-8 weeks of age) were purchased from Charles River Breeding Laboratories, Kingston, NY and Portage, MI, respectively. Animals were accl imated to laboratory conditions for at least 7 days. Animals were fed Certified Purina Chow (Ralston Purina Co., St . Louis, MO) and tap water ad libitum except during exposures. When animals were not exposed to divinylbenzene, they were placed in rooms designed to control temperature (7Z°F), relative humidity (50%) and light cycle (12 hrs light and 12 hr dark).

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: None

Details on inhalation exposure:

Chambers - Except for mice exposed to the lowest concentration of DVB-55 (0.10 mg/L), all exposures to DVB-55 were conducted in 157-liter stainless steel and glass Rochester-type chambers. Mice were exposed to 0.10 mg/L DVB-55 in 112-liter stainless steel and glass Rochester-type chambers due to a lack of small chamber availability. Chamber airflow was maintained at 30 liters/min in all chambers. The air supplied to the chambers was controlled by a system designed to maintain temperature at approximately 70°F and relative humidity at approximately 50%. The minimum and maximum temperature values during each exposure period and relative humidity at the end of each exposure period were recorded.

Test Atmosphere Generation - Groups of 5 rats/sex were exposed to 0, 0.25, 1.0 or 2.5 mg/l DVE-55 (equivalent to 0, 25, 101 or 252 ppm DVB and 0, 20, 79 or 199 ppm EVB, respectively) for 6 hrslday, 5 days/week for 9 exposures; groups of 5 micelsex were exposed to 0.10, 0.25 or 1.0 mg/l DVB-55 (equivalent to 0, 10, 25 or 101 ppm DVB and 0, 8, 20 or 79 ppm EVBy respectively) for the same time period. Vapours of divinylbenzene 55 were generated using the glass J-tube method. Liquid test material was metered at a constant rate into the glass J-tube. Heated compressed air passed through the 3-tube to volatilize the test material prior to entering the chamber. The compressed air was heated (-< 85°C) with a flameless heat torch (Master FHT-4) to the minimum extent necessary to completely vaporize the test material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration (amount of test material vaporized to the total amount of air through the chambers) was calculated for DVB-55 for each exposure period. The concentration of divinylbenzene in each chamber was determined approximately once each hour using a Varian 2400 gas chromatograph with a flame ionization detector. The air samples were separated in the gas chromatograph with a 4 ft 1/8" nickel column packed with 5% SP-1200/1.75% Bentone 34 on 100/120 Supelcoport. The conditions for the gas chromatography were as follows: Helium - 85 mL /min, air - 305 mlL/min and Hydrogen - 54 mlL/min, column temperature170°C and detector temperature 215°C. The analytical equipment was standardized by vaporizing measured volumes of the test substance in Teflon bags filled with a measured volume of air. The concentration of divinylbenzene was determined by interpolation from a standard curve via a microprocessor unit as described by Miller (1980b). In addition, at least one standard was prepared prior to each day of exposure to evaluate the analytical system.

The concentration of divinylbenzene present in the chamber was determined by gas chromatography. Although the chamber concentrations of ethylvinyl benzene were not determined analytically, ethyl vinylbenzene has a higher vapour pressure than divinylbenzene and thus would be more likely to volatilize than divinylbenzene. This was confirmed by the close agreement between analytical and nominal concentrations. Complete vaporization of 1 mg/L DVB-55 would provide a concentration of 101 ppm divinylbenzene and 79 ppm ethylvinylbenzene.

Duration of treatment / exposure:

5 days/week for 9 days

Frequency of treatment:

6 hrs/day

Doses / concentrations

Doses / concentrations 1

Remarks:

Rats (nominal concentrations) -0, 0.25, 1.0 or 2.5 mg/L (equivalent to 0, 25, 101 or 252 ppm DVB and 0, 20, 79 or 199 ppm EVB, respectively)

Doses / concentrations 2

Remarks:

Mice (nominal concentrations) - 0, 0.10, 0.25 or 1.0 mg/l (equivalent to 0, 10, 25 or 101 ppm DVB and 0, 8, 20 or 79 ppm EVB, respectively)

Doses / concentrations 3

Remarks:

Rats and mice (analytical concentrations) - 0, 12.5 ± 3.4, 32.2 ±- 6.7, 103.6 ± 7.5 or 242.4 ± 21.8 ppm

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Details on study design:

Animals were observed daily and body weights were recorded periodically. Samples were collected for haematology, clinical chemistry and urinalysis (rats only) from those animals which survived until the end of the study. A complete necropsy was performed on all animals and extensive histopathology was completed.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

Animals were observed daily and body weights were recorded periodically.

Sacrifice and pathology:

Samples were collected for haematology, clinical chemistry and urinalysis (rats only) from those animals which survived until the end of the study. A complete necropsy was performed on all animals and extensive histopathology was completed.

Other examinations:

None

Statistics:

Descriptive statistics (mean and standard deviation) were calculated for white blood cell differential counts and red blood cell indices. Body weights, absolute and relative organ weights, clinical chemistry data, appropriate haematology data and urinary specific gravity were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Statistical outliers were identified by a sequential test and excluded from analysis only for documented scientifically sound reasons unrelated to treatment. The nominal alpha levels to be used and test references were as follows:
Bartlett' s test: alpha = 0.01
Parametric AMOVA: alpha = 0.10
Non-parametric ANOVA: alpha = 0.10
Dunnett's test: alpha = 0.05, two-sided
Wilcoxon Rank-Sum test: alpha = 0.05, two-sided
Bonferroni correction
Outlier test: alpha = 0.02, two-sided
Since multiple, interrelated parameters were statistically compared in the same group of animals, the frequency of false positive errors may be much greater than the nominal alpha level. Thus, in addition to statistical analyses, the final toxicologic interpretation of the data also considered whether an orderly dose-response relationship existed and whether the findings appeared to be plausible and consistent in the light of other biologic findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mice: Exposure of mice to 1.0 mg/L DVB-55 resulted in lethargy following the initial exposure. While these animals appeared to tolerate several subsequent exposures, after the fourth exposure several mice appeared lethargic and ultimately died or were sacrificed in a moribund condition (2 of 5/sex). Mice exposed to lower concentrations of DVB-55 did not exhibit any clinical effects attributed to inhalation of DVB-55.

Rats: Exposure of rats to 2.5 mg/L DVB-55 resulted in eye irritation as manifested by closed eyelids. Rats exposed to lower concentrations of DVB-55 appeared normal throughout the exposure period.

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mice: The body weights of surviving male and female mice exposed to 1.0 mg/L DVB-55 were slightly decreased from control values during the exposure period but were not affected at lower exposure levels.

Rats: Body weights of male and female rats exposed to 2.5.mg/1 DVB-55 were slightly decreased from control values during the exposure period. Female rats in the 1.0 and 2.5 mg/L exposure groups actually lost weight between the first and fifth exposure to DVB-55. Body weights of rats exposed to 0.25 mg/L DVB-55 were comparable to control values throughout the 2-week period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Mice: Although platelet counts of male and female mice exposed to 1.0 mg/L of DVB-55 were elevated above control values, they were within the range of historical control values and thus were not considered to be of toxicologic significance. The white blood cell counts of mice exposed to 0.25 (females only) or 1.0 mg/L DVB were elevated above control values. Although WBC counts were within the range of historical control values, stress due to exposure to a high concentration of DVB-55 may have contributed to the increased number of segmented neutrophils in the survivors.

Rats: Body weights of male and female rats exposed to 2.5.mg/1 DVB-55 were slightly decreased from control values during the exposure period. Female rats in the 1.0 and 2.5 mg/L exposure groups actually lost weight between the first and fifth exposure to DVB-55. Body weights of rats exposed to 0.25 mg/L DVB-55 were comparable to control values throughout the 2-week period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mice: Several clinical chemistry and organ weight parameters in mice exposed to 1.0 mg/l were affected. These included alanine aminotransferase activity, total protein, albumin and globulin which were increased relative to control values. Serum albumin was also increased in male mice exposed to 0.25 mg/L. The alkaline phosphatase activity of female mice exposed to 1.0 mg/L was decreased from control value but was within the range of historical control values.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mice: The absolute and relative liver weights of male and female mice exposed to 1.0 mg/L DVB-55 were elevated above control values; absolute and relative thymus weights were decreased in these same animals. The decrease in thymus weights is most likely a reflection of the stressed condition of the surviving animals. The remaining absolute and relative organ weights of mice inhaling as high as 1.0 mg/L DVB-55 were interpreted as not being related to exposure to DVB-55.

Rats: At necropsy there were a number of statistically significant differences in the organ weights of rats exposed to 2.5 mg/L DVB-55. However, there were no associated gross or histopathologic changes in any of these organs. Similar organ weight changes have been reported to be a reflection of reduced body weight. Organ weights of rats exposed to lower concentrations of DVB-55 which did not result in reduced body weights, were unaffected. Thus, the organ weight changes were considered to be a secondary reflection of decreased food consumption and not directly attributed to DVB-55.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Mice: In addition to the decreased thymic mass, decreased abdominal fat was observed at the scheduled necropsy in male mice exposed to 1.0 mg/L. Male mice dying spontaneously following exposure to 1.0 mg/L DVB-55 were observed to have visceral congestion upon gross examination.

Rats: In addition to the previously noted decrease in thymic size, decreased abdominal fat was observed at the scheduled necropsy in rats exposed to 2.5 mg/L.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mice: Histopathologic examination of various tissues of animals exposed to 1.0 mg/L revealed increased basophilic staining and swelling of centrilobular hepatocytes (males only) , a decrease in lymphocytic elements of the thymic cortex (males only), and degenerative/regenerative changes in the proximal convoluted tubules of the kidneys and degenerative/regenerative changes in the olfactory neuroepithelium of the nasal tissues. The degenerative/regenerative changes in the olfactory neuroepithelium were also observed in most of the mice exposed to 0.25 mg/L DVB-55. Histopathologic examination male mice dying spontaneously following exposure to 1.0 mg/L DVB-55 revealed degenerative/regenerative changes in the olfactory neuroepithelium, necrosis of the centrilobular hepatocytes and severe necrosis of the proximal tubules of the kidneys. Results other than previously discussed effects were examined in detail and interpreted as not related to treatment.

Rats: Histopathologic examination of tissues revealed degenerative and inflammatory changes in the olfactory neuroepithelium of the nasal tissues of rats exposed to 1.0 or 2.5 mg/L DVB-55. Results other than previously discussed effects were examined in detail and interpreted as not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOEC

Remarks:

(B6C3F1 mice)

Effect level:

0.1 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: Overall effects

2

Dose descriptor:

NOAEC

Remarks:

(B6C3F1 mice)

Effect level:

0.25 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: Mice (0.25 or 1.0 mg/L) exhibited very slight/slight degenerative/regenerative alterations of olfactory neuroepithelium, which are not considered relevant for human risk assessment. Therefore, a NOAEC of 0.25 mg/L will be used for risk characterization.

3

Dose descriptor:

NOEC

Remarks:

(F344 rat)

Effect level:

0.25 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: Overall effects

4

Dose descriptor:

NOAEC

Remarks:

(F344 rat)

Effect level:

2.5 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-observable-effect level (NOEL) for DVB-55 is 0.10 and 0.25 mg/L for B6C3F1 mice and Fischer 344 rats, respectively.

Executive summary:

Mice were exposed to 0, 0.1, 0.25 or 1.0 mg/L of divinylbenzene 55 (DVB-55) for 6 hours a day, 5 days a week for nine exposures; rats were exposed to 0, 0.25, 1.0 or 2.5 mg/L of DVB-55 for the same period. Each exposure group consisted of 5 animals of each sex. Animals were observed daily and body weights were recorded periodically. Samples were collected for haematology, clinical chemistry and urinalysis ( rats only) from those animals which survived until the end of the study. A complete necropsy was performed on all animals and extensive histopathology was completed.

At the highest exposure concentration, 1.0 mg/L, four of ten mice died. Renal tubular degeneration and an increase in alanine aminotransferase activity accompanied by liver weight changes and microscopic centrilobular hepatocellular alterations were observed in this group. These morphologic changes in the liver and kidneys were most noticeable in those mice which died during exposure. Mice exposed to 0.25 or 1.0 mg/L DVB-55 also exhibited very slight to slight degenerative and regenerative alterations of the olfactory neuroepithelium.

Rats exposed to 1.0 or 2.5 mg/L DVB-55 exhibited very slight to slight degenerative and regenerative alterations of the olfactory neuroepithelium but no other histopathologic alterations. The no-observable-effect level (NOEL) for DVB-55 is 0.10 and 0.25 mg/L for B6C3F1 mice and Fischer 344 rats, respectively.