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Description of key information

A GLP-study according to OECD guideline 422 with oral gavage administration of the test material to rats is available for DVB-96. Several repeated dose inhalation studies in rats and mice, equivalent to OECD guidelines 412 and 413, are available for DVB-55 and DVB-HP. No repeated dose studies via the dermal route have been conducted with the reaction mass of divinylbenzene and ethylstyrene.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 4, 1997 – August 2, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP-study according to OECD guideline 422.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
No data
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Males, 49 days
Females, from 14 days before mating to day 3 of lactation
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Positive control:
None
Observations and examinations performed and frequency:
No data
Sacrifice and pathology:
Terminal kill: Males, day 50; Female, day 5 of lactation
Other examinations:
None
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: No effects observed.

Females: One female was moribund on day 23 of gestation during delivery
Mortality:
mortality observed, treatment-related
Description (incidence):
Males: No death and no moribundity was seen for all groups up to 1000 mg/kg/day dose.

Females: No death and no moribundity were seen for 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one death on day 17 of gestation was seen.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: No significant difference was seen in 30 and 100 mg/kg/day groups. At 300 mg/kg/day, the significantly low value was recorded on day 8 of dosing. At 1000 mg/kg/day, significantly low values were recorded during day 4 to day 50 of dosing.

Females: Before mating period, no significant difference from control group was seen at 30, 100 and 300 mg/kg/day. At 1000 mg/kg/day, significantly lower values were recorded during day 4 to day 15 of dosing. During gestation period, no significant difference from control groups was seen in 30 and 100 mg/kg/day groups. At 300 mg/kg/day, significantly low values were recorded on day 7 and 14 of gestation. At 1000 mg/kg/day, significantly low values were recorded during day 0 to day 21 of gestation. During lactation period, no significant difference from control groups was seen in 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, significantly low values were recorded on day 0 and day 4 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: At 30 mg/kg/day, no significant difference from control was seen. At 100 mg/kg/day, significantly high values were seen on day 34 and day 36. At 300 mg/kg/day, significantly high values were recorded during day 34 to day 48. At 1000 mg/kg/day, the significantly low value was seen on day 3 and the high values were recorded during day 13 to day 48.

Females: Before mating period, no significant difference from control group was seen at 30, 100 and 300 mg/kg/day. At 1000 mg/kg/day, significantly low value from control group was recorded on day 3 of dosing. During gestation period, no significant difference from control groups was seen in 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, significantly low value from control group was recorded on day 21 of gestation. During lactation period, no significant difference from control groups was seen in 30 or 100 mg/kg/day groups. At 300 mg/kg/day, significantly low value from control group was seen on day 4 of lactation. At 1000 mg/kg/day, no significant difference was recorded, but a lower tendency was observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males: At 30 mg/kg/day, a significantly high value in RBC was seen. This was not considered due to divinylbenzene dose. At 100 mg/kg/day to 300 mg/kg/day groups, no significant differences were observed for all examined items. At 1000 mg/kg/day, a slightly lower value in MCHC was recorded. As the difference was very small, this was not considered as the adverse effect of DVB dosing.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males: At 30 and 100 mg/kg/day, no significant differences were seen for all items examined. At 300 mg/kg/day, a significantly high value was recorded in beta-globulin. At 1000 mg/kg/day, significantly high values were recorded in GPT, gamma-GPT, alpha 2-globulin ratio, beta-globulin ratio and total bilirubin. Also, significantly low values were recorded in alummin, alpha 1-globulin, alpha 3-globulin and glucose.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: At 30 mg/kg/day, no significant difference from control group in absolute and relative weight was seen for all organs. At 100 mg/kg/day, a significantly high value was recorded in the absolute weight of liver. At 300 mg/kg/day, significantly high value in the absolute weight of kidneys and significantly high values in the relative weight of liver and kidneys were recorded. At 1000 mg/kg/day, significantly high values were recorded in the relative weight of liver and kidneys. Although it was not a significant difference, there was a higher tendency in the absolute weight of kidneys. In addition, significantly lower values were recorded in the absolute weight of heart, spleen and testes. Also, significantly high values were recorded in the relative weight of brain and testes. These, however, were not considered caused by divinylbenzene dosing as there was no consistent tendency in these changes.

Females: At 30 mg/kg/day, no significant difference from control group in absolute and relative weight was seen for all organs. At 100 mg/kg/day, significantly low value was recorded in the absolute weight of adrenal glands. However, this was not considered to be the adverse effects of DVB dosing because no consistent trend was found between absolute and relative weight changes. At 300 mg/kg/day, significantly high value in the relative weight of kidneys were recorded. Also, low values of heart and adrenal grands were seen. This was considered not because of DVB dosing since no consistent trend was found between absolute and relative weight changes. At 1000 mg/kg/day, significantly low values were recorded in the relative and absolute weight of thymus. Also, significantly low values of absolute spleen weight, a lower tendency of relative spleen weight and the significant high values in the absolute weight of liver, kidneys and adrenal glands were seen. In addition, significantly low values in absolute weight of brain, pituitary and heart, and the significant high values in brain and ovaries, were observed. These, however, were considered not due to DVB dosing because no consistent tendency was found between absolute and relative weight changes.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: Although slight changes were observed in liver, testis and epididymis, these changes were not considered caused by the dose of divinylbenzene because the control group showed the same changes and the number of animals with these changes were small.

Females: At 1000 mg/kg/day, there were significant differences from control group as follows: Atrophy of the thymus (7 of 10 examined), atrophy of the marginal zone in the spleen (2 of 10 examined) and degeneration/necrosis of the renal tuble of cortico- medullary junction in the kidney (3 of 10 examined).
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Critical effects observed:
no
Conclusions:
The NOAELs for repeat dose toxicity are considered to be 30 mg/kg/day for males, and 100 mg/kg/day for females.
Executive summary:

This study was conducted in accordance with OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) in male and female rats at daily oral doses of 30, 300, and 1000 mg/kg.

 

In males, no death and no moribundity were seen for all groups up to 1000 mg/kg/day dose. Significantly reduced body weights and food consumption were observed at300 mg/kg. Increased relative liver weights were observed at100 mg/kg and increased relative kidney weights were observed at300 mg/kg.

 

In females, at 1000 mg/kg, one animal died on day 17 of gestation and one was found moribund on day 23 of gestation during delivery. Suppressed body weights and food consumption were observed at300 mg/kg.

 

The NOAELs for repeat dose toxicity are considered to be 30 mg/kg/day for males, and 100 mg/kg/day for females.

Endpoint conclusion
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
no
Limit test:
no
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).
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
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)
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)
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
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.
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
Dose descriptor:
NOEC
Remarks:
(B6C3F1 mice)
Effect level:
0.1 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
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.
Dose descriptor:
NOEC
Remarks:
(F344 rat)
Effect level:
0.25 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEC
Remarks:
(F344 rat)
Effect level:
2.5 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
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.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP-study equivalent to OECD guideline 413.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female B6C3F1 mice were obtained from Taconic (Germantown, NY). On receipt, the mice were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water was available ad libitum. All animals were housed individually.
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:
Mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks.

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapour out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Buildup and decay rates for chamber vapour concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapour generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of divinylbenzene-HP in the exposure chambers were monitored by an on-line gas chromatograph equipped with FID. Samples were drawn from each exposure chamber approximately every 36 minutes.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Dose / conc.:
12.5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
No. of animals per sex per dose:
10 male and 10 female mice
Control animals:
yes
Details on study design:
Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks.
Positive control:
None
Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Core study animals were weighed initially, on day 10 or 11, weekly thereafter, and at the end of the studies.
Sacrifice and pathology:
Animals were anesthetized with carbon dioxide, and blood was collected from the supraorbital sinus of mice at the end of the study for haematology analyses. Packed cell volume; haemoglobin concentration; erythrocyte, platelet, and leukocyte counts; mean cell volume; mean cell haemoglobin; and mean cell haemoglobin concentration were determined. Manual haematocrit values were determined. A Miller disc was used to determine reticulocyte counts from blood smears. Haematology: automated and manual haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials.

Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Complete histopathology was performed on 0, 100, and 200 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The lung and nose were examined in all remaining groups of rats, and other tissues in rats were examined to a no-effect level.
Other examinations:
At the end of the studies, sperm samples were collected from male mice in the 0, 25, 50, and 100 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days during the last 2 weeks of the study from female mice in the 0, 25, 50, and 100 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lethargy or hypoactivity was observed in the higher exposure concentration groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
All 200 ppm males and nine 200 ppm females died early.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls.
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
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
histopathology: neoplastic
mortality
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
A NOAEC of 25 ppm will be taken forward for the risk characterization.
Executive summary:

Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All 200 ppm males and nine 200 ppm females died early. Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls. Lethargy or hypoactivity was observed in the higher exposure concentration groups. Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP-study equivalent to OECD guideline 413.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Taconic (Germantown, NY). On receipt, the rats were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water was available ad libitum. All animals were housed individually.
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:
Concentrations of 0, 25, 50, 100, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapour out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Buildup and decay rates for chamber vapour concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapour generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of divinylbenzene-HP in the exposure chambers were monitored by an on-line gas chromatograph equipped with FID. Samples were drawn from each exposure chamber approximately every 36 minutes.
Duration of treatment / exposure:
14 weeks
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
10/sex/dose
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Control animals:
yes
Details on study design:
Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Positive control:
None
Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Core study animals were weighed initially, on day 10 or 11, weekly thereafter, and at the end of the studies.
Sacrifice and pathology:
Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at study termination for hematology and clinical chemistry analyses. Packed cell volume; haemoglobin concentration; erythrocyte, platelet, and leukocyte counts; mean cell volume; mean cell haemoglobin; and mean cell haemoglobin concentration were determined. Manual haematocrit values were determined. A Miller disc was used to determine reticulocyte counts from blood smears. Haematology: automated and manual haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials. Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids.

Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Complete histopathology was performed on 0 and 400 ppm rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The lung and nose were examined in all remaining groups of rats, and other tissues in rats were examined to a no-effect level.
Other examinations:
At the end of the studies, sperm samples were collected from male rats in the 0, 100, 200, and 400 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days during the last 2 weeks of the study from female rats in the 0, 100, 200, and 400 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nasal/eye discharge was noted in 400 ppm males and 100 ppm females.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males.
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEC
Effect level:
50 ppm
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
no
Conclusions:
A NOEAC of 100 ppm will be taken forward for the risk characterization.
Executive summary:

Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. However, the nasal effects observed in rats after inhalation exposures are not considered relevant for human risk assessment.

Endpoint conclusion
Dose descriptor:
NOAEC
133 mg/m³
Species:
other: rats and mice
Organ:
kidney
liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
no
Limit test:
no
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).
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
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)
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)
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
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.
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
Dose descriptor:
NOEC
Remarks:
(B6C3F1 mice)
Effect level:
0.1 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
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.
Dose descriptor:
NOEC
Remarks:
(F344 rat)
Effect level:
0.25 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEC
Remarks:
(F344 rat)
Effect level:
2.5 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
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.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP-study equivalent to OECD guideline 413.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female B6C3F1 mice were obtained from Taconic (Germantown, NY). On receipt, the mice were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water was available ad libitum. All animals were housed individually.
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:
Mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks.

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapour out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Buildup and decay rates for chamber vapour concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapour generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of divinylbenzene-HP in the exposure chambers were monitored by an on-line gas chromatograph equipped with FID. Samples were drawn from each exposure chamber approximately every 36 minutes.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Dose / conc.:
12.5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
No. of animals per sex per dose:
10 male and 10 female mice
Control animals:
yes
Details on study design:
Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks.
Positive control:
None
Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Core study animals were weighed initially, on day 10 or 11, weekly thereafter, and at the end of the studies.
Sacrifice and pathology:
Animals were anesthetized with carbon dioxide, and blood was collected from the supraorbital sinus of mice at the end of the study for haematology analyses. Packed cell volume; haemoglobin concentration; erythrocyte, platelet, and leukocyte counts; mean cell volume; mean cell haemoglobin; and mean cell haemoglobin concentration were determined. Manual haematocrit values were determined. A Miller disc was used to determine reticulocyte counts from blood smears. Haematology: automated and manual haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials.

Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Complete histopathology was performed on 0, 100, and 200 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The lung and nose were examined in all remaining groups of rats, and other tissues in rats were examined to a no-effect level.
Other examinations:
At the end of the studies, sperm samples were collected from male mice in the 0, 25, 50, and 100 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days during the last 2 weeks of the study from female mice in the 0, 25, 50, and 100 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Lethargy or hypoactivity was observed in the higher exposure concentration groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
All 200 ppm males and nine 200 ppm females died early.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls.
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
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
histopathology: neoplastic
mortality
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
A NOAEC of 25 ppm will be taken forward for the risk characterization.
Executive summary:

Groups of 10 male and 10 female mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All 200 ppm males and nine 200 ppm females died early. Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls. Lethargy or hypoactivity was observed in the higher exposure concentration groups. Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP-study equivalent to OECD guideline 413.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Taconic (Germantown, NY). On receipt, the rats were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water was available ad libitum. All animals were housed individually.
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:
Concentrations of 0, 25, 50, 100, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapour out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Buildup and decay rates for chamber vapour concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapour generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of divinylbenzene-HP in the exposure chambers were monitored by an on-line gas chromatograph equipped with FID. Samples were drawn from each exposure chamber approximately every 36 minutes.
Duration of treatment / exposure:
14 weeks
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
10/sex/dose
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Control animals:
yes
Details on study design:
Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Positive control:
None
Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Core study animals were weighed initially, on day 10 or 11, weekly thereafter, and at the end of the studies.
Sacrifice and pathology:
Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at study termination for hematology and clinical chemistry analyses. Packed cell volume; haemoglobin concentration; erythrocyte, platelet, and leukocyte counts; mean cell volume; mean cell haemoglobin; and mean cell haemoglobin concentration were determined. Manual haematocrit values were determined. A Miller disc was used to determine reticulocyte counts from blood smears. Haematology: automated and manual haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials. Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids.

Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Complete histopathology was performed on 0 and 400 ppm rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (bronchial, mandibular, mediastinal, mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The lung and nose were examined in all remaining groups of rats, and other tissues in rats were examined to a no-effect level.
Other examinations:
At the end of the studies, sperm samples were collected from male rats in the 0, 100, 200, and 400 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days during the last 2 weeks of the study from female rats in the 0, 100, 200, and 400 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nasal/eye discharge was noted in 400 ppm males and 100 ppm females.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males.
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased.
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEC
Effect level:
50 ppm
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
no
Conclusions:
A NOEAC of 100 ppm will be taken forward for the risk characterization.
Executive summary:

Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. However, the nasal effects observed in rats after inhalation exposures are not considered relevant for human risk assessment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route: A combined repeated-dose toxicity and reproductive and developmental toxicity study (OECD 422) has been conducted using oral gavage administration of divinylbenzene (96%) to male and female rats. Rats were exposed to 0, 30, 100, 300, or 1000 mg/kg bw day. There were no deaths or moribund cases in any of the male groups. However, there was one death and one moribund case in the high-dose female group. In the high dose group, lowered skin temperatures, depilation and soiling of fur were seen for both sexes. In addition, reduced spontaneous movements and bleeding from the vagina was seen in the females. Both males and females showed reduced body weight gain and reduced food consumption at the two high dose levels. The males also showed changes in blood chemistry. High Increased relative liver weights were seen in males at 100 mg/kg, and increased absolute and relative liver weights, or tendencies towards this, were seen in the 300 mg/kg and higher groups. For females, high relative weights of the kidneys were seen in the 300 mg/kg and 1000 mg/kg group. Reduced relative and absolute thymus weights, low absolute spleen weights, and a tendency towards low relative spleen weight, as well as increased relative liver, kidney, and adrenal weights were seen in the 1000 mg/kg group. No histopathological changes were observed in any of the males. In females, atrophy of both the cortex and medulla of the thymus, atrophy of the marginal zone of the spleen and degeneration / necrosis of the urinary tubules in the cortex-medulla boundary of the kidneys were seen in the high dose group. In the moribund cases central necrosis in the lobules of the liver, reduction of thymogen granules in the acinar cells of the pancreas, erosion of the glandular stomach mucosa and reduced hematopoiesis of the bone marrow were seen. The NOEL of DVB-96 based on general toxicological effects was 30 mg/kg/day for the males (due to high relative livers weights seen from the 100 mg/kg dose) and 100 mg/kg for the females (due to suppression of body weight increases and a decrease in food consumption from the 300 mg/kg dose).

Inhalation route: Several studies via the inhalation route are available for DVB-HP and DVB-55.

In a 14-day inhalation study with DVB-55, rats and mice were exposed daily to 0.25, 1.0 and 2.5 mg/L or 0.1, 0.25 and 1.0 mg/L, respectively. Rats exposed to 1.0 or 2.5 mg/L of DVB-55 exhibited very slight to slight degenerative and regenerative alterations of olfactory neuroepithelium, but no other histopathologic alterations. The nasal effects are not considered relevant for human risk assessment based on read-across data from structurally related chemicals (see below). Therefore, a NOAEC of 2.5 mg/L (470 ppm) will be taken forward for the risk characterization. Four out of ten mice died at the highest concentration (1.0 mg/L). 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 of DVB-55 also exhibited very slight to slight degenerative and regenerative alterations of the olfactory neuroepithelium. The nasal effects are not considered relevant for human risk assessment. Therefore, a NOAEC of 0.25 mg/L (47 ppm) will be taken forward for the risk characterization.

In a 13-week inhalation study in rats with daily exposure to 25, 50, 100, 200 and 400 ppm of DVB-HP all rats survived to the end of the study. At the high dose levels increased weights of kidney, liver, heart and testis were observed. The increase in kidney weights in male rats is considered to be a species-specific effect in male rats due to alpha-2-µ globulin mediated effects. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. In another 13-week inhalation study with mice and exposure concentrations of 12.5, 25, 50, 100 and 200 ppm of DVB-HP all high-dose males and nine females died early. Final mean body weights were significantly lower in males and females exposed to 25, 50, or 100 ppm when compared with chamber controls. Lethargy or hypoactivity was observed in the higher exposure concentration groups. Exposure to divinylbenzene was associated with necrosis of the liver and kidney in 200 ppm males and females dying early. In all exposed groups of male and female mice, there was necrosis of nasal cavity lateral walls, olfactory epithelium, and glands with resultant atrophy of olfactory epithelium and glands in females. A lower number of animals had necrotic or degenerative changes of the upper respiratory tract. However, the nasal effects observed in rats and mice after inhalation exposure to DVB-HP are not considered relevant for human risk assessment based on read-across data from structurally related chemicals (see below). Therefore, a NOAEC of 100 ppm for rats and a NOAEC of 25 ppm for mice will be taken forward for the risk characterization. In mice the only other effect observed at 25 ppm was a decrease in final body weights which can be considered secondary to the nasal irritation and general discomfort as it is not accompanied by any systemic effect at this dose level. Overall similar effects were observed in the 14-day inhalation studies with DVB-HP in rats and mice at higher dose levels resulting in a NOAEC of 100 ppm for rats and 50 ppm for mice. Based on the similarity of the effects observed in the 14-day inhalation studies with DVB-55 and DVB-HP at comparable dose levels, it can be concluded that the NOAEC from the sub-chronic studies with DVB-HP are also applicable to and sufficiently protective for exposure to DVB-55 and DVB-63.

Available information on treatment-related histopathologic nasal effects in rats and mice following exposure to DVB or styrene for 3 months or 2 years, were reviewed to determine if these effects were comparable. In mice, inhalation of DVB at 25 ppm or greater for 3 months was reported to cause necrosis of olfactory epithelium, which was not reported in mice after 3-month exposure to styrene even at 200 ppm (exception: 2 early deaths at 200 ppm styrene were reported to have manifested marked olfactory necrosis). It must be noted that DVB inhalation study was conducted in B6C3F1 mice and styrene studies were conducted with CD-1 mice and whether or not this strain difference may have contributed to this discrepancy is not clear. With the exception of some minor differences, all other nasal effects induced by DVB were comparable to those induced by styrene, although comparison to similar dose-levels could not be made (lowest doses for DVB and styrene were 12.5 and 50 ppm respectively). Comparison of the histopathologic nasal effects induced by DVB or styrene inhalation in mice for a 2-year period showed, that with the exception of suppurative inflammation in the nasal lumen of mice treated with DVB at 10 ppm or greater, other reported nasal effects also occurred with styrene exposure and thus, in general, were comparable to the spectrum of changes described for styrene induced nasal effects in mice. It is concluded from the review of available information, that the described nasal effects for rats and mice exposed to DVB, in general, were comparable to most, if not all effects described for styrene. However, the important difference noted was the occurrence of necrosis of olfactory epithelium in mice exposed to DVB for 3 months at concentrations of 25 ppm or above, an effect not described in mice following 3-month exposure to styrene even at the highest dose of 200 ppm. However, at the end of 2 years, nasal effects were largely comparable between those caused by DVB or styrene. Although, the exact reasons for this difference noted at the end of 3-months are not clear, it may be due to a higher irritancy and/or other possible olfactory metabolic effects of DVB compared to those of styrene.

Further, it has been established that rats and mice are particularly sensitive to respiratory tract toxicity following styrene exposure. Inhalation of styrene by mice results in cytotoxicity in terminal bronchioles, followed by increased incidence of bronchioloalveolar tumors, as well as degeneration and atrophy of nasal olfactory epithelium. In rats, no effects on terminal bronchioles are seen, but effects in the nasal olfactory epithelium do occur, although to a lesser degree and from higher exposure concentrations. Whole-body metabolism studies have indicated major differences in styrene metabolism between rats and mice. The major differences are 4- to 10-fold more ring-oxidation and phenylacetaldehyde pathways in mice compared to rats. The data indicate that local metabolism of styrene is responsible for cytotoxicity in the respiratory tract. Cytotoxicity is seen in tissues that are high in CYP2F P450 isoforms. These tissues have been demonstrated to produce a high ratio of R-SO compared to S-SO (at least 2.4:1). In other rat tissues the ratio is less than 1, while in mouse liver the ratio is about 1.1. Inhibition of CYP2F with 5-phenyl-1-pentyne prevents the styrene-induced cytotoxicity in mouse terminal bronchioles and nasal olfactory epithelium. RSO has been shown to be more toxic to mouse terminal bronchioles than S-SO. In addition, 4-vinylphenol (ring oxidation of styrene) has been shown to be highly toxic to mouse terminal bronchioles and is also metabolized by CYP2F. In human nasal and lung tissues, styrene metabolism to SO is below the limit of detection in nearly all samples, and the most active sample of lung was approximately 100-fold less active than mouse lung tissue. It can be concluded that styrene respiratory tract toxicity in mice and rats, including mouse lung tumors, are mediated by CYP2F-generated metabolites. The PBPK model predicts that humans do not generate sufficient levels of these metabolites in the terminal bronchioles to reach a toxic level. Therefore, the postulated mode of action for these effects indicates that respiratory tract effects in rodents are not relevant for human risk assessment.

According to the assessment presented above the nasal effects caused by inhalation exposure to DVB are comparable to the effects caused by inhalation exposure to styrene. In addition, it was shown that the major routes of DVB hepatic metabolism are comparable to styrene and it is expected that the routes of non-hepatic metabolism of DVB would also be consistent with those observed for styrene (see section 7.1.1 of IUCLID). Therefore, it is concluded that the nasal effects observed in mice and rats after inhalation exposure to DVB are not relevant for human exposure to DVB.

Justification for classification or non-classification

The NOAECs from the repeated dose inhalation studies are above the threshold for classification and the only effect observed after repeated oral exposure to 100 mg/kg bw/day of DVB-HP was relative increased liver weights (without accompanying histopathology) which is considered to be an adaptive effect. Therefore, the reaction mass of divinylbenzene and ethylstyrene does not need to be classified for repeat dose toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.