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PubMed1986

NTP Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C23, 43% Chlorine) (CAS No. 108171-27-3) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

Toxicology and carcinogenesis studies of chlorinated paraffins (C23, 43% chlorine), an extreme-pressure lubricant and flame retardant, were conducted by administering the chemical in corn oil by gavage to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days per week for 103 weeks. Additional groups of 10 rats per sex and dose were examined at 6 and at 12 months. Male rats received doses of 0, 1,875, or 3,750 mg/kg body weight; female rats were given 0, 100, 300, or 900 mg/kg. Male and female mice received 0, 2,500, or 5,000 mg/kg. Doses selected for the 2-year studies were based on the results from 13-week studies in which rats of each sex received 0 to 3,750 mg/kg, and mice of each sex, 0 to 7,500 mg/kg. No toxicity of chlorinated paraffins (C23, 43% chlorine) was observed in male rats or in male or female mice in the 13-week studies. A dose-related inflammation of the liver was observed in female rats in the 13-week studies and in male and female rats in the 13-week studies and in male and female rats at 6 and 12 months in the 2-year studies. Chlorinated paraffins (C23, 43% chlorine) administration did not influence mean body weights of rats during the 2-year studies, but both male and female low dose mice gained less weight than did vehicle controls or the high dose groups. Survival of dosed and vehicle control groups was similar for each sex and species (male rats: vehicle control, 30/50; low dose, 32/50; high dose, 27/50; female rats: 34/50; 30/50; 33/50; 31/50; male mice: 29/50; 36/50; 28/50; female mice: 21/50; 22/50; 20/50). For female mice, 60%-70% of the early deaths in each group were attributed to utero-ovarian infection. The lower survival for female mice may have decreased the sensitivity of this study to detect a carcinogenic effect. Pheochromocytomas of the adrenal gland medulla occurred with an increased incidence in female rats exposed to chlorinated paraffins (C23, 43% chlorine) (vehicle control, 1/50; low dose, 4/50; mid dose, 6/50; high dose, 7/50). However, adrenal gland medullary hyperplasia was not increased (6/50; 3/50; 1/50; 6/50). Malignant lymphomas were increased in dosed male mice (6/50; 12/50; 16/50). High dose female mice showed a marginal increase in the incidence of hepatocellular carcinomas (1/50; 1/49; 6/50) and in the incidence of adenomas or carcinomas (combined) (4/50; 3/49; 10/50). The primary nonneoplastic lesion associated with chlorinated paraffins (C23, 43% chlorine) administration was a diffuse lymphohistiocytic inflammation in the liver and in the pancreatic and mesenteric lymph nodes of male and female rats. Splenic congestion was a secondary effect. These lesions occurred earlier and at lower doses in female rats than in male rats. No significant nonneoplastic lesions were considered compound related in mice. Chlorinated paraffins (C23, 43% chlorine) was not mutagenic in strains TA100, TA1535, TA97, or TA98 of Salmonella typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-Dawley rat or male Syrian hamster liver S9 when assayed according to the preincubation protocol. An audit of the experimental data was conducted for these 2-year studies of chlorinated paraffins (C23, 43% chlorine). No data discrepancies were found that influenced the final interpretations. Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for male F344/N rats given 1,875 or 3,750 mg/kg per day. There was equivocal evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for female F344/N rats as shown by an increased incidence of adrenal gland medullary pheochromocytomas. There was clear evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for male B6C3F1 mice as shown by an increase in the incidence of malignant lymphomas. There was equivocal evidence of carcinogenicity of chlorinated paraffins (C23, 43% chlorine) for female B6C3F1 mice as shown by a marginal increase in the incidence of hepatocellular neoplasms. *The Chemical Abstract Service Seal increase in the incidence of hepatocellular neoplasms. *The Chemical Abstract Service Service (CAS) number that appeared on this technical report at the time of publication (63449-39-8) reflects the generic CAS number for chlorinated paraffins. This number has been replaced in the NTP Chemtrack chemical tracking system with the more appropriate number.

Journal Article↗

PubMedDate not supplied

Immunohistochemical detection of P-glycoprotein on frozen and paraffin-embedded tissue sections of normal and malignant tissues.

In this study the reactivity of the monoclonal antibodies (mabs) C 219, C 494 and 4E3 was compared on frozen and paraffin-embedded normal tissues and tumors. On frozen tissues we used an indirect immunoperoxidase method, while on paraffin sections a streptavidin-biotin method without antigen retrieval methods was used. In normal tissues all mabs were reactive with colon epithelium in frozen and paraffin-embedded sections. The bile canaliculi in the liver showed the most extensive reaction with C 219 in frozen sections, and to a lesser extent with C 494 and 4E3. C 219 reacted with the pancreatic acinar and ductal epithelium, whereas C 494 and 4E3 reacted predominantly in the stroma. The kidney showed positivity for all mabs in the collecting ducts and isolated solitary cells were reactive in the spleen. In the skin the eccrine part of the sweat glands was reactive for all mabs in paraffin sections. The lung, prostate and breast were negative for all mabs. Only in paraffin sections of various tissues did the C 494 appear to be reactive with nerve fibers and ganglion cells. Colon cancers were positive for P-170 with all mabs tested. In breast carcinoma C 494 showed positive reactions in 8/26 frozen and 4/22 paraffin sections, while 4E3 was reactive with 20/25 frozen but only with 1/21 paraffin sections. C 219 gave similar results in frozen (22/26) and paraffin (17/26) sections of breast cancer. Ovarian carcinomas were positive with C 494 in 11/20 of the frozen and in 11/15 of the paraffin sections, while 4E3 again reacted more weakly in paraffin (5/15) than in frozen (15/20) sections. C 219 gave positive reactions in all ovarian carcinomas in frozen (20/20) and paraffin sections (14/14). In the tumors, the most intense reaction for all mabs was obtained in the colon, followed by the ovary and breast. Enhanced staining was seen in paraffin sections for mab C 494 in ovarian carcinoma. By demonstrating the presence of both an external and internal epitope on frozen sections, the combined use of 4E3 and C 219 gave complementary information about the expression of P-170.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Controlled release of insect sex pheromones from paraffin wax and emulsions.

Paraffin wax and aqueous paraffin emulsions can be used as controlled release carriers for insect sex pheromones for mating disruption of orchard pests. Paraffin can be applied at ambient temperature as an aqueous emulsion, adheres to tree bark or foliage, releases pheromone for an extended period of time, and will slowly erode from bark and biodegrade in soil. Pheromone emulsions can be applied with simple spray equipment. Pheromone release-rates from paraffin were measured in laboratory flow-cell experiments. Pheromone was trapped from an air stream with an adsorbent, eluted periodically, and quantified by gas chromatography. Pheromone release from paraffin was partition-controlled, providing a constant (zero-order) release rate. A typical paraffin emulsion consisted of 30% paraffin, 4% pheromone, 4% soy oil, 1% vitamin E, 2% emulsifier, and the balance water. Soy oil and vitamin E acted as volatility suppressants. A constant release of oriental fruit moth pheromone from paraffin emulsions was observed in the laboratory for more than 100 days at 27 degreesC, with release-rates ranging from 0.4 to 2 mg/day, depending on the concentration and surface area of the dried emulsion. The use of paraffin emulsions is a viable method for direct application of insect pheromones for mating disruption. Sprayable formulations can be designed to release insect pheromones to the environment at a rate necessary for insect control by mating disruption. At temperatures below 38 degreesC, zero-order release was observed. At 38 degreesC and higher, pheromone oxidation occurred. A partition-controlled release mechanism was supported by a zero-order pheromone release-rate, low air/wax partition coefficients, and pheromone solubility in paraffin.

Air↗

PubMed1987

Comparative toxicity and carcinogenicity of two chlorinated paraffins in F344/N rats and B6C3F1 mice.

The toxicity and carcinogenicity of chlorinated paraffins containing C12 with 60% Cl, and C23 with 43% Cl, were assessed in prechronic and 2-year gavage studies using F344/N rats and B6C3F1 mice of both sexes. Single administrations of chlorinated paraffins were nonlethal in rats and mice, but repeated-dose and 2-year studies demonstrated toxic responses that differed with the paraffins. The C23,Cl43% paraffin produced a granulomatous inflammation in the liver of female rats in 13-week studies, while the C12,Cl60% paraffin caused deaths of rats and mice in 16-day studies and marked liver enlargement in 13-week studies. In 2-year studies, the C23,Cl43% paraffin caused hepatic and lymphatic granulomatous inflammation and hyperplasia in both sexes of rats, and was associated with marginal increases in adrenal medullary pheochromocytomas in female rats and hepatocellular neoplasms in female mice and with clear increases in malignant lymphomas in male mice. The C12,Cl60% paraffin caused marked liver enlargement in rats and increased the severity of nephropathy in male rats and the incidence of nephropathy in female rats. C12,Cl60% also caused hepatocellular neoplasms in both sexes of rats and mice: kidney tubular cell adenomas and adenocarcinomas in male rats, thyroid follicular cell neoplasms in female rats and female mice, and a marginal increase in mononuclear cell leukemia in male rats. Thus, the short-chain, heavily chlorinated paraffin appears to have a greater potential for chronic toxicity and carcinogenicity than the longer-chain, lightly chlorinated paraffin. Both paraffins have been reported to be nonmutagenic in bacteria. (National Toxicology Program (1986) Technical Report, NIH Publications 86-2561 and 86-2564).

Animals↗

PubMed2004

Hepatocyte paraffin 1 antibody does not distinguish primary ovarian tumors with hepatoid differentiation from metastatic hepatocellular carcinoma.

Anti-hepatocyte antibody, hepatocyte paraffin 1, is a monoclonal antibody that is highly specific for normal and neoplastic hepatocytes and that can differentiate hepatocytic from nonhepatocytic tumors. This marker has been rarely studied in extra-hepatic tumors and to our knowledge has not been investigated in ovarian tumors with hepatoid differentiation. We studied hepatocyte paraffin 1 immunoreactivity in a series of ovarian hepatoid carcinomas, ovarian hepatoid yolk sac tumors (YSTs), and hepatocellular carcinomas metastatic to the ovary to assess the potential utility of hepatocyte paraffin 1 in differential diagnosis. Hepatocyte paraffin 1 positivity was seen in three of seven ovarian hepatoid carcinomas, five of eight hepatoid yolk sac tumors, and six of eight metastatic hepatocellular carcinomas. The extent of positivity ranged from <25% to >50% of the tumor cells. There was strong coarsely granular cytoplasmic staining in all three tumor types without a distinctive staining pattern in any group. The degree of hepatic differentiation correlated with hepatocyte paraffin 1 positivity in the three groups: 83% of the well differentiated tumors, 50% of the moderately differentiated tumors, and none of the poorly differentiated tumors were positive. All ovarian hepatoid carcinomas were either immunoreactive for alpha-fetoprotein or had an elevated serum alpha-fetoprotein level; more than half of these tumors were hepatocyte paraffin 1 negative. All but one hepatocyte paraffin 1 negative hepatoid yolk sac tumor and ovarian hepatocellular carcinoma were also negative for alpha-fetoprotein. In conclusion, hepatocyte paraffin 1 is positive in primary ovarian tumors with hepatoid differentiation, with the degree of hepatocyte paraffin 1 positivity correlating with the degree of hepatoid differentiation. Hepatocyte paraffin 1, however, is not useful in distinguishing metastatic hepatocellular carcinoma from primary ovarian hepatoid carcinoma or hepatoid yolk sac tumor.

Adolescent↗

PubMed1968

Further evidence for an elongation-decarboxylation mechanism in the biosynthesis of paraffins in leaves.

In isolated tobacco leaves l-valine-U-(14)C gave rise to labeled even-numbered isobranched fatty acids containing 16 to 26 carbon atoms and iso C(29), iso C(31), and iso C(33) paraffins. l-Isoleucine-U-(14)C on the other hand produced labeled odd-numbered anteiso C(17) to C(27) fatty acids and anteiso C(30) and C(32) paraffins. Trichloroacetic acid inhibited the incorporation of isobutyrate into C(20) and higher fatty acids and paraffins without affecting the synthesis of the C(16) and C(18) fatty acids. Thus the very long branched fatty acids are biosynthetically related to the paraffins. In Senecio odoris leaves acetate-1-(14)C was incorporated into the paraffins (mainly n-C(31)) only in the epidermis although acetate was readily incorporated into fatty acids in the mesophyll tissue. Similarly only the epidermal tissue incorporated acetate into fatty acids longer than C(18) suggesting that the epidermis is the site of synthesis of both paraffins and the very long fatty acids. In broccoli leaves n-C(12) acid labeled with (14)C in the carboxyl carbon and (3)H in the methylene carbons was incorporated into C(29) paraffin without the loss of (14)C relative to (3)H. Since n-C(18) acid is known to be incorporated into the paraffin without loss of carboxyl carbon these results suggest that the condensation of C(12) acid with C(18) acid is not responsible for n-C(29) paraffin synthesis in this tissue. Thus all the experimental evidence thus far obtained strongly suggests that elongation of fatty acids followed by decarboxylation is the most likely pathway for paraffin biosynthesis in leaves.

Journal Article↗

PubMed2005

A comparative study: the efficacy of liquid paraffin and lactulose in management of chronic functional constipation.

OBJECTIVES: To determine and compare efficacy, safety and optimal dose of two laxatives, liquid paraffin and lactulose, in 40 children with chronic functional constipation. METHODS: A total of 20 children were treated with liquid paraffin and 20 with lactulose for 8 weeks and at an initial dose of 1 mL/kg per day for both drugs. The dose was adjusted every 3 days as required and a diary was kept to monitor dose, side-effects, stool frequency and consistency, and other symptoms. RESULTS: During first 4 weeks, improvement in stool consistency and frequency was significantly higher in liquid the paraffin group (P < 0.01 and P < 0.05, respectively). Improvement in the number of stools per week was also significantly higher in the liquid paraffin group during the last 4 weeks of therapy (P < 0.05). Compliance rates averaged 95% in the liquid paraffin group and 90% in the lactulose group during the first 4 weeks of therapy and 90% in the liquid paraffin group and 60% in the lactulose group during the last 4 weeks of therapy (chi(2) = 4.8, SD = 1, P = 0.02). During the first 4 weeks of therapy and during the last 4 weeks of therapy, mostly side-effects and poor symptom control, respectively, influenced the compliance in the liquid paraffin group. CONCLUSIONS: Liquid paraffin is more effective in the treatment of children with constipation. Patients treated with liquid paraffin responded more rapidly than patients responding to lactulose and showed fewer side-effect. It is considered that late response and side-effects in conjunction with poor symptom control influence the patient compliance and the successful treatment of childhood constipation.

Cathartics↗

PubMed1998

Lack of asbestos contamination of paraffin.

OBJECTIVE: To investigate possible asbestos contamination of paraffin and migration by asbestos fibers during the tissue-embedding process. DESIGN: Three sample categories were included in the study: (1) commercially available paraffin samples; (2) procedural control samples, which were prepared by processing the paraffin through the use of standard solvents and instruments; and (3) samples taken from areas adjacent to embedded tissue and evaluated for migration of asbestos from the tissue into the surrounding paraffin. The analysis of collected material from all samples was performed with analytical transmission electron microscopy. RESULTS: Only one extremely small tremolite fiber was found in any of the commercially available samples of paraffin. No asbestos fibers were found either in the procedural control samples or in the samples taken adjacent to the embedded lung tissue. CONCLUSIONS: First, it was extremely unlikely that any of the commercial paraffin samples would have skewed data due to embedded tissue. Second, the processing and instrumentation was not found to contribute asbestos material to the paraffin during the preparations. Finally, embedded tissue that contained high numbers of fibers, both uncoated fibers and asbestos bodies, did not contribute asbestos to the adjacent paraffin.

Asbestos↗

PubMed1996

Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections.

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin-paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a 'test battery' approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120 degrees C, 100 degrees C, and 90 degrees C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100 degrees C proved most effective. Both fresh and routinely processed formalin-paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin-paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin-paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.

Cryopreservation↗

PubMed1990

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses.

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.

Adult↗

PubMed2002

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.

Antigens↗

PubMed1997

[DNA flow cytometry. Comparison of the methods for nuclear extraction from frozen and paraffin-embedded sections].

Cellular DNA content of solid tumors can be determined either from fresh, frozen, or formalin fixed, paraffin-embedded tissues. However, discordant results have been obtained using the paraffin-embedded technique, and lack of abnormal DNA stemlines in the paraffin-embedded as compared to either fresh or frozen tissues has been reported. In this study we evaluated the validity of nuclear extraction method from paraffin-embedded tissues, using 75 breast carcinomas whose DNA content was previously analyzed from frozen tissue and resulted either normal (12 cases) or abnormal (63 cases). From representative paraffin blocks, nuclei were extracted following Hedley's technique. The results revealed excellent cell counting and good histogram resolution from all paraffin samples; the loss of G2M abnormal peak in eight histograms with abnormal stemline did not compromise the correct interpretation of DNA content. In addition, the comparison between DNA indices obtained from corresponding paraffin and frozen samples showed a good correlation in 69 cases (r = 94); discordance in six cases was demonstrated to be related to tumor heterogeneity. In conclusion the paraffin extraction method is a sensible, and reliable technique, which can be applied for DNA flow cytometric studies on archival cases, as well as whenever fresh sample from the tumor is not obtainable.

Aneuploidy↗

PubMed1968

Species specificity in the biosynthesis of branched paraffins in leaves.

Isobutyrate-1-(14)C and l-isoleucine-U-(14)C fed through the petiole labeled the surface lipids of broccoli leaves, but the incorporation was much less than from straight chain precursors. Not more than one-third of the (14)C incorporated into the surface lipids was found in the C(29) paraffin and derivatives, whereas more than two-thirds of the (14)C from straight chain precursors are usually found in these compounds. The small amount of (14)C incorporated into the paraffin fraction was found in the n-C(29) paraffin rather than branched paraffins showing that the (14)C in the paraffin must have come from degradation products. Radio gas-liquid chromatography of the saturated fatty acids showed that, in addition to the n-C(16) acid which was formed from both branched precursors, isoleucine-U-(14)C gave rise to branched C(15), C(17), and C(19) fatty acids, and isobutyrate-1-(14)C gave rise to branched C(16) and C(18) acids. Thus the reason for the failure of broccoli leaf to incorporate branched precursors into branched paraffins is not the unavailability of branched fatty acids, but the absolute specificity of the system that synthesizes paraffins, probably the elongation-decar-boxylation enzyme complex. Consistent with this view, no labeled branched fatty acids longer than C(19) could be found in the broccoli leaf. The branched fatty acids were also found in the surface lipids indicating that the epidermal layer of cells did have access to branched chains. Thus the paraffin synthesizing enzyme system is specific for straight chains in broccoli, but the fatty acid synthetase is not.

Journal Article↗

PubMed1986

NTP Toxicology and Carcinogenesis Studies of Chlorinated Paraffins (C12, 60% Chlorine) (CAS No. 108171-26-2*) in F344/N Rats and B6C3F1 Mice (Gavage Studies).

Toxicology and carcinogenesis assessments of chlorinated paraffins (C12, 60% chlorine), a material widely used as a flame retardant and extreme-pressure lubricant, were conducted in male and female F344/N rats and male and female B6C3F1 mice in single-administration, 16-day, 13-week, and 2-year studies. Doses used in the 2-year studies were 0, 312, or 625 mg/kg body weight per day administered by gavage in corn oil five times per week to groups of 70 male and female rats and 0, 125, or 250 mg/kg administered to groups of 50 male and female mice. Ten male and 10 female rats were killed after 6 and 12 months of dosing and examined for toxicity. No chemically related toxicity was observed in single-administration studies in which male and female rats received doses of chlorinated paraffins (C12, 60% chlorine) up to 13,600 mg/kg body weight and male and female up to 27,200 mg/kg. In 16-day studies, deaths did occur in groups of male and female rats given 7,500 mg/kg and in groups of male and female mice given doses of 1,875 mg/kg or higher. In 13-week studies, no chemically related deaths occurred among male and female rats given up to 5,000 mg/kg or mice given up to 2,000 mg/kg. Increased liver weights were noted in dosed rats and mice of each sex in the short-term studies, and dosed male rats showed more severe nephropathy than did vehicle controls. Doses selected for the 2-year studies were those that caused a minimal increase in liver weight in the short-term studies. Liver and kidney weights were increased in dosed rats killed at 6 and 12 months. Morphometric measurements demonstrated hepatocyte hypertrophy in the livers of dosed rats. Lesions of the kidney tubules and interstitial inflammation increased with dose in male and female rats. During the 2-year studies, body weights of high dose male rats were 8%-12% lower than those of vehicle controls after week 20, and body weights of dosed female mice were about 10% lower than those of vehicle controls during the second year. Survival of dosed male rats was lower than that of vehicle controls after about week 85, perhaps due to toxicity to the kidney (final survival: vehicle control, 27/50; low dose, 6/50; high dose, 3/50). Survival of low dose female rats was lower than that of vehicle controls (34/50; 24/50; 29/50). Survival of dosed male mice was not significantly different from that of vehicle controls (34/50; 31/50; 31/50). Survival of high dose female mice was lower than that of vehicle controls after about week 75 (final survival: 36/50; 31/50; 25/50). Chemically related nonneoplastic lesions consisted of hypertrophy and minimal focal necrosis of the liver in rats; erosion, inflammation, and ulceration of the glandular stomach and forestomach a in male rats; and formation ofmultiple cysts in the kidney tubules of male rats. The incidence of nephropathy was also increased in dosed female rats and mice. The maximum tolerated dose may have been exceeded in male and female rats. Neoplastic lesions associated with chlorinated paraffins (C12, 60% chlorine) administration were found in the liver of rats and mice of each sex (see table p. 12 of Technical Report) Dosed male rats showed increased incidences of kidney tubular cell hyperplasia (1/50; 9/50; 12/49) and of tubular cell adenomas (0/50; 7/50; 3/49); two low dose males had tubular cell adenocarcinomas. The incidences of mononuclear cell leukemia were increased in dosed male rats (7/50; 12/50; 14/50) and in low dose female rats (11/50; 22/50; 16/50). Pancreatic acinar cell tumors occurred at increased incidences in low dose male rats (11/50; 22/50; 17/50). Follicular cell adenomas or carcinomas (combined) of the thyroid gland were found at increased incidences in both female rats (0/50; 6/50; 6/50) and female mice (8/50; 12/49; 15/49). Chlorinated paraffins (C12, 60% chlorine) was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 in the presence or absence of Aroclor 1254-induced male Sprague-Dawley or male Syrian hamster liver S9 when tested according to the preincubational protocol. An ed according to the preincubational protocol. An audit of the experimental data was conducted for these 2-year studies on chlorinated paraffins (C12, 60&percnt; chlorine). No data discrepancies were found that influenced the final interpretations. Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenicity of chlorinated paraffins (C12, 60&percnt; chlorine) for F344/N rats based on increased incidences of hepatocellular neoplasms (primarily neoplastic nodules) in male and female rats, of adenomas or adenocarcinomas (combined) of the kidney tubular cells in male rats, and of follicular cell adenomas or carcinomas (combined) of the thyroid gland in female rats. Mononuclear cell leukemia in dosed male rats may have been related to administration of chlorinated paraffins (C12, 60&percnt; chlorine). There was clear evidence of carcinogenicity of chlorinated paraffins (C12, 60&percnt; chlorine) for B6C3F1 mice as shown by increased incidences of hepatocellular adenomas and of adenomas or carcinomas (combined) in dosed male and female mice and increased incidences of adenomas and of adenomas or carcinomas (combined) of thyroid gland follicular cells in dosed female mice.

Journal Article↗

PubMed1994

The impact of child-resistant containers on the incidence of paraffin (kerosene) ingestion in children.

The commonest cause of accidental poisoning in the South African black paediatric population is paraffin ingestion. In this intervention study a specifically designed child-resistant container (CRC) was introduced to evaluate whether its use would decrease the incidence of paraffin ingestion. CRCs were distributed to 20,000 households in the study area (Gelukspan district). No CRCs were distributed in the control area (Lehurutshe district). Health education about paraffin poisoning prevention was given in both the control and the study areas. The monthly incidence rates of paraffin ingestion were monitored during the 14-month intervention period after the distribution and were compared with the pre-intervention incidence rates in the study and control areas. The main finding was that the incidence of paraffin ingestion dropped by 47% in the study area during the intervention period. The circumstances surrounding the cases of paraffin ingestion that still occurred in the study and control areas were investigated by means of a questionnaire. We recommend that paraffin be sold in CRCs, and suggestions are made for improving health education to prevent paraffin poisoning.

Accidents, Home↗

PubMed2003

Estimation of hormone receptor status in fine-needle aspirates and paraffin-embedded sections from breast cancer using the novel rabbit monoclonal antibodies SP1 and SP2.

We describe a method of immunocytochemical assessment of estrogen receptor (ER) status on alcohol-fixed smears obtained by fine-needle aspiration (FNA) from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-ER (SP1) without any antigen retrieval. A series of 40 aspirates were analyzed and the results of ER status were compared with the respective formalin-fixed tissue using the same procedure and with assessment by the classical method using the mouse monoclonal antibody 6F11 (anti-ER) with antigen retrieval on paraffin sections. Twenty-four out of the 40 cases examined were positive at least by two methods and 16 were negative for all three determinations. The results obtained in the ER immunocytochemical assay on aspirates and paraffin sections using the antibody SP1 and those obtained on paraffin sections using the antibody 6F11 were quite similar. In one case the material was insufficient to interpret the reaction in the cytological specimen and only one case, with focal positivity reaction on paraffin sections, was negative in the cytological specimen. The intensity of nuclei staining in cytological smears of breast cancer cells was stronger than that observed by traditional methods. We also assessed progesterone receptor (PR) status on 40 paraffin-sections from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-PR (SP2), with the same characteristics described for anti-ER (SP1). The results were compared with assessment by the classic method with mouse monoclonal antibody 1A6 (PR) on paraffin sections and total agreement was observed. Of the 40 cases examined, 18 were positive and 22 were negative for the two determinations. We conclude that the application of the ER method on alcohol-fixed smears/paraffin sections with the rabbit monoclonal antibody SP1, and the PR method on paraffin sections with the rabbit monoclonal antibody SP2, provide several advantages, such as high sensitivity and specificity of the reaction, stronger immunostaining, shorter procedures times, and avoidance of antigenic retrieval methods.

Adenocarcinoma↗

PubMed2002

Southwestern histochemistry as a molecular histochemical tool for analysis of expression of transcription factors: application to paraffin-embedded tissue sections.

Southwestern histochemistry is used to localize transcription regulatory factors that bind to specific sequences of DNA and regulate the transcriptional activity of the genes, using a haptenized double-stranded DNA. This method has been developed in fresh frozen sections, but it was difficult to detect a specific signal in paraffin sections. In this review, we focus on the use of Southwestern histochemistry in paraffin sections. To detect a reliable signal in paraffin sections, we autoclaved or microwaved the sample before incubation with probe DNA. As a model system, we assessed estrogen receptor (ER) in paraffin sections of mouse ovary. ER was detected by Southwestern histochemistry in autoclaved or microwaved paraffin sections, and no signal was detected without such pretreatment. The ER staining pattern detected by Southwestern histochemistry was well consistent with that of ER alpha and ER beta detected by immunohistochemistry. Next, we analyzed the expression of cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) in regenerating livers of rats and mice. CREB was detected in hepatocyte nuclei in autoclaved paraffin sections by Southwestern histochemistry. Immunohistochemistry of CREB displayed homogeneous staining in hepatocyte nuclei, while the signal detected by Southwestern histochemistry showed a heterogeneous speckled pattern in the nuclei. CREB expression increased prior to the induction of proliferating cell nuclear antigen (PCNA). Because the PCNA gene has a cAMP-responsive element in its promoter, it can be suggested that CREB may activate the PCNA gene and lead to the induction of hepatocyte DNA synthesis. Our results indicate that autoclave or microwave treatment is very useful for Southwestern histochemistry in paraffin-embedded sections.

Animals↗

PubMed1995

Discordant CD3 expression in lymphomas when studied on frozen and paraffin sections.

The results of CD3 staining in the T or putative natural killer (NK) cell lymphomas of nasal and extranasal sites as reported in the literature have been confusing, with some studies reporting a low rate of CD3 positivity and others a high frequency of CD3 staining. The former studies were performed on fresh or frozen tissues, whereas the latter were performed on paraffin sections using a polyclonal antiserum (poly-CD3). Although previous studies have suggested a high concordance rate of CD3 staining between fresh/frozen tissues and paraffin sections, many CD3- cases have not been studied, and the more reliable antigen retrieval techniques have not been applied. In this study, we addressed the question of discordant CD3 expression by comparing the results of CD3 staining in lymphomas as studied on frozen sections and as studied on paraffin sections (with antigen retrieval effected by pressure cooking). This series was biased toward inclusion of a high percentage of cases of putative NK cell lymphomas, which are prevalent among Asians and usually show a CD2+ CD3(Leu4)- CD56+ immunophenotype. Among 35 cases of CD3(Leu4)- T- and T/NK-cell lymphomas, 30 (86%) showed staining with poly-CD3 on paraffin sections. All 15 CD3(Leu4)+ T-cell lymphomas showed positive staining with poly-CD3 on paraffin sections. None of 60 B-cell lymphomas were stained by poly-CD3, confirming no loss of specificity of staining with this antiserum despite use of an effective antigen-retrieval technique. The discordance rate of CD3 staining in T- and T/NK-cell lymphomas in this series was 60%, and this phenomenon was most commonly observed in the CD56+ T/NK-cell lymphomas: CD3(Leu4)- in frozen sections but poly-CD3+ in paraffin sections. Therefore, to avoid confusion, we propose designating the results based on fresh/frozen tissues CD3(f) and those based on poly-CD3 application on paraffin sections CD3(p) in future reporting of CD3 immunophenotype.

Antigens, CD↗