(1) A 21-day chronic Daphnia magna reproduction toxicity study was conducted by Thompson et al. (1997b) according to OECD 202, Part II (1984) using a static-renewal test system with renewal 3 times/week. The test substance was identified as Cereclor S52[(R)], a C14-17 chlorinated paraffin with 52% chlorination that contained 0.3% epoxy soya bean oil stabilizer as well as a small amount of radiolabeled n-pentadecane-8-[14]C (51% chlorinated). Ten replicates of 1 Daphnia magna Straus (<24 hours old) were tested per exposure concentration, which did not comply with OECD 202, Part II requirements that at least 40 daphnid be tested per concentration. Nominal concentrations were 0 (dilution water control), 0 (solvent control), 0.0056, 0.01, 0.018, 0.032, 0.056, and 0.1 mg/L test substance in acetone (0.025 mL/L). Results from the acute daphnid study by the same author do not appear to have been considered when selecting concentrations for this study. Test solutions were prepared by adding the appropriate stock solution to dilution water while continuously and vigorously stirring with a magnetic follower. The submitter does not indicate whether renewal of the static-renewal test systems was carried out at regular intervals (e.g, Monday-Wednesday-Friday). Corresponding mean measured concentrations determined by radiochemical methods were 0.0037, 0.005, 0.01, 0.018, 0.032, and 0.065 mg/L and were 78-94% of nominal concentrations at the start of the renewal period and 7.3-61% of nominal concentrations at the end of the renewal period indicating a notable loss of test substance. Also, analysis of test concentrations appears to be at irregular intervals. In the dilution water control, 20% mortality was observed. Overall, dilution water control and solvent control results were significantly different for reproductive parameters. The test was carried out at temperatures of 19.5-20.3 °C, at pH levels of 7.41-8.13, and at dissolved oxygen concentrations of 6.2-9.2 mg/L. A significant decrease in the number of live offspring was reported at the mean measured concentration of 0.018 mg/L and delayed release of first offspring was observed at higher concentrations. Percentage dead offspring reported was 0%, 0%, 5.9%, 20.4%, and 18.5% for the 0.0037, 0.005, 0.01, 0.018, 0.032, and 0.065 mg/L mean measured exposures.

                  EPA/OPPT Conclusion
                  The inability to maintain test concentrations, unspecified renewal periods, and a smaller population size may have affected subsequent reproductive results. Given the uncertainties of the test, reported effect levels may not represent a worst case scenario but do exhibit a clear dose response relationship with a clearly defined statistically significant effect level. Thus, using a weight of evidence approach, the study is considered acceptable for this endpoint.
                  21-day LC50 value of 0.025 mg/L (parent mortality)
                  21-d NOEC = 0.01 mg/L
                  21-d LOEC = 0.018 mg/L
                  21-d GMATC = 0.013 mg/L

(2) A 60-day mussel toxicity study was conducted by Brixham Laboratories in 1983 with radio-labeled chlorinated (52%) n-pentadecane (Trade Name: Cereclor S52[(R)]) under flow through testing conditions. A full non-CBI study report was submitted under TSCA in 1983 as DCN 40-8332184 (OTS Fiche 0507258). Blue mussels (Mytilus edulis) were exposed to nominal concentrations of 0 (dilution sea water control), 0 (acetone control, 500 ppm), 0.56, or 5.6 mg/L in 500 ppm acetone. Two replicates of 50 mussels were tested for the dilution water and solvent controls and a single replicate of 50 mussels was exposed for each treatment concentration. Corresponding mean measured concentrations were 0, 0, 0.22, and 3.8 mg/L. Test solutions were cloudy at the higher test concentration. Test concentrations were determined by radio activity measurements. Flow rate of the test system was 0.25 mL/minute for exposure concentrations. Over the course of the study, water temperature ranged from 14.6  -  15.6 °C, pH ranged from 8.0  -  8.3, and the dissolved oxygen concentrations ranged from 6.1  -  8.25 mg/L. Dilution water salinity was 34-35.5 ppb, which is high by OCSPP standards.

                  EPA/OPPT Conclusion
                  One mussel exposed to 0.56 mg/L died, and two mussels exposed to the controls died; this was not considered to be a test substance related effect. Decreases in filter feeding were observed at 5.6 mg/L. In addition, the submitter provided an assessment of bioconcentration, but this assessment does not appear to include a depuration phase. Overall, the 60 day NOEC and LOEC were 0.22 and 3.8 mg/L based on reduced filtration. The study was acceptable to characterize mussel toxicity, but mussels are not considered a standard species to fulfill the chronic aquatic invertebrate toxicity endpoint.
                  60-d NOEC = 0.22 mg/L
                  60-d LOEC = 3.8 mg/L (reduced filtration)

(3) The following study summary (Frank, 1993; Frank and Steinhäuser, 1994) provided in the 2005 European Chemical Bureau Risk Assessment of MCCP was considered supportive of the aquatic invertebrate hazard determination. A 21-day chronic Daphnia magna reproduction toxicity study was conducted using a static-renewal test system (renewal 3 times/week). The test substance was identified as C14-17 chlorinated paraffin with 52% chlorination, which was tested as a water soluble fraction of two stock solutions (dilutions used 1:2 to 1:32). Nominal test concentrations prepared from the 100 mg/L stock solution were 3.125, 6.25, 12.5, 25, and 50 mg/L. Nominal test concentrations prepared from the 10,000 mg/L stock solution were 312.5, 625, 1250, 2500, and 5000 mg/L. Analytical monitoring of test concentrations was conducted, but only the final effect levels were presented as measured concentrations. Methods for test solution preparation were not provided in the summary. The tests were carried out at 20 °C and at  pH 7.79-8.44.

In the experiments using the 100 mg/L stock solution the mortality seen in the exposed populations was 0% at 3.125 mg/L, 0% at 6.25 mg/L, 20% at 12.5 mg/L, 90% at 25 mg/L, and 100% at 50 mg/L. In the experiments using the 10,000 mg/L stock solution the mortality seen in the exposed populations was 0% at 312.5 mg/L, 30% at 625 mg/L, 70% at 1250 mg/L, and 100% at lower dilutions (>1250 mg/L). In the experiments using the 100 mg/L stock solution the average number of young/adult was 82 at 3.125 mg/L, 89 at 6.25 mg/L, 80 at 12.5 mg/L, 15 at 25 mg/L and 0 at 50 mg/L (all parents died). Similarly in the experiments using the 10,000 mg/L stock solution the average number of young/adult was 74 at 312.5 mg/L, 64 at 625 mg/L, 43 at 1250 mg/L, and 0 at 2,500 and 5,000 mg/L (all parents died). Based on these effects, survivability/mortality appears to be the more sensitive endpoint. Based on the known measured concentrations in the stock solutions and the dilution rates used the NOEC for mortality was around 0.0044-0.0089 mg/L for the 100 mg/L nominal stock solution experiments and 0.0126-0.0156 mg/L for the 10000 mg/L nominal stock solution experiments. The corresponding LOECs were 0.0089- 0.0178 mg/L (100 mg/L nominal stock) and 0.0253-0.0313 μg/L (10 g/L nominal stock). The GMATC of 0.006 mg/L was calculated using the geometric mean from the most conservative NOEC (0.0044 mg/L) and LOEC (0.0089 mg/).

                  EPA/OPPT Conclusion
                  EPA/OPPT reserves judgment on the acceptability of this study until further details become available.
                  21-d NOEC = 0.0044 mg/L
                  21-d LOEC = 0.0089 mg/L
                  21-d GMATC = 0.006 mg/L

3) The following study summary (TNO, 1993) provided in the 2005 European Chemical Bureau Risk Assessment of MCCP was considered supportive of the aquatic invertebrate hazard determination. A 21-day chronic Daphnia magna reproduction toxicity study was conducted using a static-renewal test system (renewal 3 times/week). The test substance was identified as C14-17 chlorinated paraffin with 52% chlorination. The test solutions were prepared by stirring 20 g of the test substance in 2 litres of heated water (60 °C) with stirring and then filtration through a 0.8 um and 0.2 um filter. This resulting stock solution was referred to as a water soluble fraction, but given that each concentration was not independently prepared, the test solutions is considered by EPA/OPPT to be merely a mixed and filtered solution that was subsequently diluted. Following dilution of the stock solution, exposure concentrations were analytically determined using the extractable organic halogen method, but not provided in the study summary. The test was carried out at 20 +- 1 ºC and solutions were gently aerated from day 9 onwards. The pH of the test water varied between 7.7 and 8.3, the dissolved oxygen concentration was > 7 mg/L, and the hardness was 214 mg CaCO3/L. Test solutions were clear.

                  EPA/OPPT Conclusion
                  Although analytical results obtained were considered to be too erratic to allow precise determination of concentrations (according to the study summary in ECB, 2005), the NOEC was reported based on survivability and/or reproductive effects. A LOEC in mg/L was not reported in the summary, nor could EPA/OPPT extrapolate one. EPA/OPPT reserves judgment on the acceptability of this study until further details become available.
                  21-d NOEC = 0.004-0.008 mg/L

 Chronic Aquatic Sediment Invertebrate Toxicity
1) Thompson et al. (2001c) conducted a 28-day prolonged sediment invertebrate toxicity study with spiked sediment was conducted according to OECD 218 draft guideline (February 2000 version) using a static test system. The substance used in the test was commercial C14-17, 52% wt. Cl substance containing no stabilizers (the substance was reported to have a C14-17 content of 99.06% with 0.67% of C10-13 chain length substances) that was mixed with a small amount of a radio labeled n-pentadecane-8-[14]C, 51% wt. Cl substance (radiochemical purity >96.6%). The sediment used in the test was an artificial sediment that did not fully adhere to the final OECD TG recommendations, but the composition of 10% sphagnum moss peat, 70% quartz sand, 20% kaolinite clay, and <0.1% calcium carbonate are not considered to be significantly different. The sediment had a mean organic carbon content of 4.9% and a pH of 6.0. The sediment was spiked with the test substance by firstly mixing a solution of the test substance in acetone with the dry sand component of the sediment and allowing the acetone to evaporate overnight under an air stream. Measured concentrations were determined using radiochemical analysis. Over the course of the study, temperature was maintained at 20 ºC, pH levels were 6.2-7.6, and dissolved oxygen in overlying water was maintained at 7.3-8.6 mg/L. Three replicates of 15 midge (Chironomus riparius) larvae (<48 hours post hatch) were exposed to mean measured concentrations of 0 (sediment control), 0 (solvent sediment control), 36, 110, 370, 1200, 3800, or 13000 mg/kg dry wt. sediment. Time to first emergence, mean emergence time, mean number emerged per replicate, and sex ratio was assessed for each exposure group. Statistically significant effects (p = 0.05) were limited to a decrease in mean number emerged per replicated in the 13,000 mg/kg dry wt. sediment exposed midges.

                  EPA/OPPT Conclusion
                  The overall NOEC of 3,800 mg/kg dry wt. sediment corresponded to 1,460 mg/kg on a wet weight basis. The study is acceptable.
                  28-d NOEC = 3,800 mg/kg dry wt sediment
                  28-d LOEC = 13,000 mg/kg dry wt sediment
                  28-d GMATC = 7,029 mg/kg dry wt sediment

2) Thompson et al. (2001d) conducted a 28-day prolonged sediment invertebrate toxicity study with spiked sediment according to methods described in Phipps et al. (1993) using a static test system. The substance used in the test was commercial C14-17, 52% wt. Cl substance containing no stabilizers (the substance was reported to have a C14-17 content of 99.06% with 0.67% of C10-13 chain length substances) that was mixed with a small amount of a radio labelled n-pentadecane-8-14C, 51% wt. Cl substance (radiochemical purity >96.6%). The sediment used in the test was an artificial sediment consisting of 10% sphagnum moss peat, 70% quartz sand, 20% kaolinite clay, and <0.1% calcium carbonate. The sediment had a mean organic carbon content of 4.9% and a pH of 6.0. The test sediments were made up by adding the test substance to the sand phase as a solution in acetone, evaporating the acetone overnight and mixing the spiked sand with the rest of the sediment for 16 hours. Six replicates of 10 oligochaete (Lumbriculus variegatus) adults were exposed to mean measured concentrations of 0 (sediment control), 0 (solvent sediment control), 39, 130, 410, 1300, 4000, or 13000 mg/kg dry wt. sediment. Throughout the duration of the study, water temperature was maintained at 20 +- 1 Cº, and pH remained between 6.3 and 7.9. Mortality and reproductive success were determined by total number of worms at study termination since differentiation of adult and young worms is difficult. Mean number of worms per replicate and mean total dry weight of worms per replicate was significantly different from controls (p = 0.01) at mean measured concentrations of 410 mg/kg dry weight sediment and greater. Statistical methods used were not reported, and a p-value of 0.01 was used.

                  EPA/OPPT Conclusion
                  Given that a clear decline in the mean number of worms per replicate and mean total dry weight of worms per replicate was observed at the lowest test concentration (39 mg/kg dry weight sediment) a conservative LOEC of 39 mg/kg dry weight sediment will be used based on noticeable differences. EPA/OPPT reserves judgment on the acceptability of this study until further details become available regarding the analytical measurements of the chlorinated paraffin mixture.
                  28-d NOEC = 130 mg/kg dry wt sediment
                  28-d LOEC = 410 mg/kg dry wt sediment
                  GMATC = 230.9 mg/kg dry wt sediment

3) Thompson et al. (2002) conducted a 28-day prolonged sediment toxicity study with amphipod Hyalella azteca in spiked sediment using a static-renewal test system with weekly renewals. The substance used in the test was a mixture of a commercial medium-chain chlorinated paraffin product (C14-17, 52.5% wt. Cl) mixed with a small amount of a radiolabeled chlorinated n-pentadecane-8-14C (51% wt. Cl). The sediment used in the test was an artificial sediment consisting of 10% sphagnum moss peat, 70% quartz sand, 20% kaolinite clay, and <0.1% calcium carbonate. The sediment had a mean organic carbon content of 4.9% and a pH of 6.0. The test sediments were made up by adding the test substance to the sand phase as a solution in acetone, evaporating the acetone overnight and mixing the spiked sand with the rest of the sediment and water. Six replicates per concentration of ten juvenile Hyalella azteca (~7-day-old) were exposed to 0 (sediment control), 0 (acetone sediment control), 38, 75, 150, 300, or 600 mg/kg dry weight sediment. The concentration of the test substance was measured in the sediment phase by radiochemical analysis with concentrations at the start of the exposure period of 85-97% of the nominal values and concentrations at the end of the 29-day exposure period of 78-90% of nominal. Results of the test were expressed as the arithmetic mean concentration. Over the course of the study, dissolved oxygen ranged from 7.7 to 8.4 mg/L, pH ranged from 7.0 to 7.6, water hardness ranged from 41 to 42 mg CaCO3/L, and temperature ranged from 22.4-23.2ºC. The endpoints investigated in the study included survival, growth (dry weight) and sexual development of females (proportion of gravid females).

                  EPA/OPPT Conclusion
                  Controls responded adequately. For the survival endpoint, a statistically significant (p=0.05) reduction in survival was seen at 470 mg/kg dry weight. A statistically significant reduction in mean weight was seen in females only at exposures of 470 g/kg dry weight sediment and a statistically significant (p=0.05) reduction in mean weight was seen at 270 mg/kg dry weight. For the sexual development endpoint, there was a statistically significant (p=0.05) reduction in the proportion of gravid females in the 470 mg/kg dry weight treatment. This study was acceptable.
                   28-d NOEC = 130 mg/kg dry wt sediment
                  28-d LOEC = 270 mg/kg dry wt sediment
                  28-d GMATC = 187 mg/kg dry wt sediment

 Avian Toxicity
(1) An acute avian toxicity study conducted according to OPPTS guidelines was published by Madeley & Birtley (1980). Following a range-finding study, groups of 5 male and 5 female ring-necked pheasants (Phasianus colchicus) were exposed by gavage to 0 (control) or 24,606 mg/kg Cereclor S52[(R)]  (C14-17, 52% Cl) and then observed for 14 days. Based on reported tissue concentrations, the test substance is believed to have been absorbed by the ring-necked pheasant. Doses up to 24,606 mg/kg failed to produce any abnormal clinical signs or mortality.

                  EPA/OPPT Conclusion
                  Acute LD50 > 24,606 ppm

(2) An acute avian toxicity study conducted according to OCSPP guidelines was published by Madeley & Birtley (1980). Following a range-finding study, groups of 5 male and 5 female mallard ducks (Anas platyrynchos) were exposed by gavage to 0 (control) or 10,280 mg/kg Cereclor S52[(R)]  (C14-17, 52% Cl) and then observed for 14 days. Based on reported tissue concentrations, the test substance is believed to have been absorbed by the mallard ducks. Doses up to 10,280 mg/kg failed to produce any abnormal clinical signs or mortality.

                  EPA/OPPT Conclusion
                  Acute LD50 > 10,280 ppm

(3) A sub-acute dietary avian toxicity study conducted according to OPPTS guidelines was published by Madeley & Birtley (1980). Following a range-finding study, groups of 5 male and 5 female ring-necked pheasants (Phasianus colchicus) were exposed to diets containing 0 (control), 1,000, or 24,063 ppm Cereclor S52[(R)]  (C14-17, 52% Cl) for 5 days. Three groups were exposed to the negative control and two groups were exposed to each of the treatment concentrations. Based on reported tissue concentrations, the test substance is believed to have been absorbed by the ring-necked pheasant. Good health was noted in all control and treatment groups. No abnormal effects were noted at necropsy.

                  EPA/OPPT Conclusion
                  5-day LD50 > 24,063 ppm

(3) A sub-acute dietary avian toxicity study conducted according to OPPTS guidelines was published by Madeley & Birtley (1980). Following a range-finding study, groups of 5 male and 5 female mallard ducks (Anas platyrynchos) were exposed to diets containing 0 (control), 1,000, or 24,063 ppm Cereclor S52[(R)]  (C14-17, 52% Cl) for 5 days. Three groups were exposed to the negative control and two groups were exposed to each of the treatment concentrations. Inferior food intake was noted for ducks, but weight gain was comparable to controls. Based on reported tissue concentrations, the test substance is believed to have been absorbed by the mallard ducks. Good health was noted in all control and treatment groups. No abnormal effects were noted at necropsy.

                  EPA/OPPT Conclusion
                  5-day LD50 > 24,063 ppm
 Terrestrial Invertebrate Toxicity
1) A 28-day earthworm reproductive toxicity test was conducted by Thompson et al., 2001a according to OECD guideline (2000 draft version). The substance tested was commercial C14-17, 52% wt. Cl substance containing no stabilizers (the substance was reported to have a C14-17 content of 99.06% with 0.67% of C10-13 chain length substances) and a small amount of 14C-labelled n-pentadecane, 51% wt. Cl substance. Four replicates per concentration of 10 adult earthworm (Eisenia fetida) were exposed to nominal concentrations of 0 (soil control), 0 (solvent soil control), 100, 320, 1000, 3200, or 10,000 mg/kg dry wt. soil. Corresponding mean measured concentrations of 0 (soil control), 0 (solvent soil control), 79, ~280, 900, ~2,800, or 9,300 mg/kg dry wt. was determined using radiochemical analysis; concentrations identified as approximate (~) were approximated using the mean% of nominal (87%) determined in other treatments. Measured tissue concentrations in adults on day 28 were 169, 802, and 732 mg/kg wet weight for the 79, 900, and 9,300 mg/kg dry weight exposure groups. Measured tissue concentrations in juveniles on day 56 were 140 and 1,011 mg/kg wet weight for the 79 and 900 mg/kg dry weight exposure groups. The soil used in the test was an artificial soil consisting of 10% sphagnum moss peat, 70% quartz sand, 20% kaolinite clay, and 0.25% calcium carbonate. The soil had an organic carbon content of 4.7% and a pH of 6.66-7.09. Nominal test temperatures remained at 20 +- 1 ºC. The soils were prepared up by firstly adding the test substance in solution with acetone to a small portion of soil, evaporating out the acetone overnight under a stream of compressed air, and then mixing with the remainder of the soil. Before use, distilled water was added to the dry soil to provide a soil wet:dry ratio of 1.35. Following the 28-day parental exposure period, adult earthworms were removed, and vessels were incubated for an additional 28 days to allow hatching of any egg cocoons produced by parent. Effects assessed were parental survival, growth as determined by change in weight of parents, and reproduction as determined by number of live offspring. A statistically significant (p = 0.05) reduction in parental survival (85%) was observed at 9,300 mg/kg dry wt. soil. A statistically significant (recalculated with Dunnett's Procedure, P = 0.05) reduction in parental weight was reported at 2800 mg/kg dry wt. soil. A statistically significant (recalculated with Dunnett's Procedure, P = 0.05) reduction in number of live offspring was reported at 280 mg/kg dry wt. soil. In addition, the submitter assesses corresponding tissue concentrations in earthworms and determines that at nominal concentrations of 100 mg/kg dry wt. soil the concentration in parental earthworm tissue after 28 days is 850 mg/kg dry wt.and in juvenile worms after 56 days was 703 mg/kg dry wt.

                  EPA/OPPT Conclusion
                  The study was acceptable.
                  28-d NOEC = 79 mg/kg dry wt soil
                  28-d LOEC = 280 mg/kg dry wt soil
                  28-d ChV = 149 mg/kg dry wt soil

2) The following study summary (Thompson et al., 2001a) provided in the 2005 European Chemical Bureau Risk Assessment of MCCP was used to characterize terrestrial invertebrate hazard in a screening level risk assessment. A 28-day earthworm reproductive toxicity test was conducted according to OECD guidelines. The substance tested was commercial C14-17, 52% wt. Cl substance containing no stabilizers (the substance was reported to have a C14-17 content of 99.06% with 0.67% of C10-13 chain length substances). The test substance contained a small amount of 14C-labelled n-pentadecane, 51% wt. Cl substance so that radiochemical analysis could be used to analytically determine test concentrations in soil. The soil used in the test was an artificial soil consisting of 10% sphagnum moss peat, 70% quartz sand, 20% kaolinite clay, and 0.25% calcium carbonate. The soil had an organic carbon content of 4.7% and a pH of 6.66-7.09. The soils were prepared up by firstly adding the test substance in solution with acetone to a small portion of soil, evaporating out the acetone overnight under a stream of compressed air, and then mixing with the remainder of the soil. Before use, distilled water was added to the dry soil to provide a soil wet:dry ratio of 1.35. Four replicates per concentration of 10 adult earthworm (Eisenia fetida) were exposed to mean measured concentrations of 0 (soil control), 0 (solvent soil control), 79, ~280, 900, ~2800, or 9300 mg/kg dry wt. for 28 days; concentrations identified as approximate (~) were approximated using the mean% of nominal (87%) determined in other treatments. Measured tissue concentrations in adults on day 28 were 169, 802, and 732 mg/kg wet weight for the 79, 900, and 9300 mg/kg dry weight exposure groups. Measured tissue concentrations in juveniles on day 56 were 140 and 1011 mg/kg wet weight for the 79 and 900 mg/kg dry weight exposure groups. Following the 28-day parental exposure period, adult earthworm were removed, and vessels were incubated for an additional 28 days to allow hatching of any egg cocoons produced by parent. Effects assessed were parental survival, growth as determined by change in weight of parents, and reproduction as determined by number of live offspring. Statistical methods used to calculate significance were not provided. A statistically significant (p = 0.05) reduction in parental survival (85%) was observed at 9300 mg/kg dry wt. soil. A statistically significant (p = 0.01) reduction in parental weight was reported at 2800 mg/kg dry wt. soil and a noticeable reduction in parental weight was reported at 280 mg/kg dry wt. soil. A statistically significant (p = 0.01) number of live offspring was reported at 1000 mg/kg dry wt. soil and a noticeable reduction in number of live offspring was reported at 320 mg/kg dry wt. soil. A clear decline in parental weight and number of live offspring was observed at 280 mg/kg dry wt. soil.

                  EPA/OPPT Conclusion
                  This study is acceptable.
                  28-d NOEC = 79 mg/kg dry wt soil
                  28-d LOEC = 280 mg/kg dry wt soil
                  28-d GMATC = 149 mg/kg dry wt soil

 Terrestrial Plant Toxicity
1) Thompson et al., 2001ab conducted a 28-day seed germintation and vegetative vigor study. The toxicity of a C14-17, 52% wt. Cl substance (99.06% purity) to wheat (Triticum aestivum; monocotyledon), oilseed rape (Brassica napus; ditcotyledon), and mung bean (Phaseolus aureus; dicotyledonous legume) has been studied using OECD guideline 208 (July, 2000 Revision). The test substance contained a small amount of 14C-labelled n-pentadecane, 51% wt. Cl substance so that radiochemical analysis could be used to analytically determine test concentrations in soil. The soils were prepared up by firstly adding the test substance in solution with acetone to dry silver sand, evaporating the acetone overnight, and mixing the spiked sand with the soil. Four replicate pots per exposure concentration each containing 9 seeds were exposed for 28 days to nominal exposure concentrations of 0 (soil control), 0 (solvent soil control), 50, 158, 500, 1,580, or 5,000 mg/kg dry wt.. According to the E.U. Risk Assessment, use of nominal test concentrations to determine effect levels is based on test substance stability determined at the 50, 500, and 5,000 mg/kg dry wt. concentrations (corresponding mean measured concentrations: 49, 520, and 5,800 mg/kg dry wt.). Effects assessed were seed germination, emergence (% emerged plants on Day 14), vegetative growth (mean shoot dry weight per plant), and visual appearance of seedling. No statistically significant differences (p = 0.05) were observed in wheat, oilseed rape. A statistically significant reduction in growth was seen at 1,580 and 5,000 mg/kg dry wt. for mungbean when compared to soil control results.

                  EPA/OPPT Conclusion
                  Since soil control and solvent control means were equal (two tailed T-Test) indicating no solvent interference, comparison of treatments was made to the soil control. Thus, the NOEC and LOEC for terrestrial plants was 500 and 1,580 mg/kg dry wt. soil and the GMATC (geometric mean of the NOEC and LOEC) was 888.8 mg/kg dry wt. soil. The study was acceptable to characterize both monocot (wheat) and dicot (mung bean) seed germination and vegetative vigor; reproductive effects remain uncharacterized.
                  28-d NOEC = 500 mg/kg dry wt soil
                  28-d LOEC = 1,580 mg/kg dry wt soil
                  28-d GMATC = 888.8 mg/kg dry wt soil

 Conclusions
Sufficient data were available to characterize the acute fish, the acute aquatic invertebrate, the chronic aquatic invertebrate, the chronic aquatic sediment invertebrate, avian, and terrestrial plant toxicity endpoints for MCCPs. Data for other toxicity endpoints (i.e., chronic fish, aquatic plant, etc.) were inconclusive due to lack of study details, uncertainties in analytical methods, or test material preparation methods; thus, these data are included in order to characterize risk in a qualitative manner, but are used as supportive for the categories under which they are provided. Supporting data were included in order to provide a weight-of-evidence approach used to characterize some endpoints.

Most of the data provided in this review indicated several difficulties were encountered when testing in an aquatic environment. These included: (1) getting the material into solution, (2) measuring the material in solution, and (3) characterizing the effects for each study listed. Often there were many details of a given study omitted, prohibiting a full and robust review of the data. The (estimated) physical-chemical properties of MCCPs (water solubility values of approximately 30 μg/L and Log Kow values between 4-8) suggest these materials may not partition to the aquatic media or elicit toxicity to aquatic organisms within the water column.

The most reliable and acceptable studies indicate that for MCCPs, the toxicity to aquatic organisms for acute endpoints are from the Thompson et al. 1996 study for aquatic invertebrates. Where the 48-hour EC50 value = 0.0059 mg/L. Using the methods described in the Sustainable Futures/P2 Manual (US EPA, 2012), the acute and chronic concentrations of concern (CoC) are derived as follows:
 Acute CoC: The 48-hour EC50 value = 0.0059 mg/L is divided by an assessment factor of 5 to yield an acute concentration of concern (CoC) of 0.00118 mg/L, or 0.001 mg/L, or 1 μg/L (1 ppb). Aquatic Acute Concern Concentration= 1 ppb
 Chronic CoC: The aquatic invertebrate chronic value of 0.013 mg/L from the 1997 Thompson et al. study based on a MCCP material is divided by an assessment factor of 10 to yield 0.0013 mg/L or 1.3 μg/L or 1.3 ppb. Aquatic Chronic Concern Concentration = 1 ppb

The most reliable and acceptable value for the toxicity to aquatic sediment invertebrate organisms acute endpoint is based on the MCCP material from the Thompson et al. 2002 28-d study. The 28-d sediment invertebrate GMATC value of 187 mg/kg dry wt sediment is used to assess hazard. Again, using methods in US EPA (2012):

 Acute CoC: Calculating an acute concern concentration from the chronic value of 187 mg/kg dry wt. The 187 value is multiplied by an acute to chronic ratio for invertebrates (10) to yield 1,870 mg/kg dry wt. This value is then divided by an assessment factor of 5 to yield 374 mg/kg dry wt. Aquatic Sediment Acute Concern Concentration = 374 mg/kg dry wt sediment.
 Chronic CoC: The 28-d sediment invertebrate GMATC of 187 mg/kg dry wt sediment is divided by an assessment factor of 10 to yield 18.7 mg/kg dry wt sediment. Aquatic Sediment Chronic Concern Concentration = 19 mg/kg dry wt sediment.

The most reliable and acceptable value for the toxicity to terrestrial invertebrates acute endpoint is based on the MCCP material from the Thompson et al. 2001a study. The 28-d terrestrial invertebrate GMATC value of 149 mg/kg dry wt sediment from this study will be used.
Again, using methods in US EPA (2012):

 Acute CoC: Calculating an acute concern concentration from the chronic value of149 mg/kg dry wt, this value is multiplied by an acute to chronic ratio for invertebrates (10) to yield 1,490 mg/kg dry wt. This value is then divided by an assessment factor of 5 to yield 298 mg/kg dry wt. Terrestrial Invertebrate Acute Concern Concentration = 298 mg/kg dry wt sediment.
 Chronic CoC: The 28-d terrestrial invertebrate GMATC of 149 mg/kg dry wt is divided by an assessment factor of 10 to yield 14.9 mg/kg dry wt sediment. Terrestrial Invertebrate Chronic Concern Concentration = 15 mg/kg dry wt sediment.

The most reliable and acceptable value for the toxicity to terrestrial plants is based on the MCCP material from the Thompson et al. 2001ab study. For LCCPs, the analog approach using the values from this study may be used. However, there is no OPPT guidance regarding assessing concern concentrations for terrestrial plants.

 LCCP ECOTOXICITY DATA
Where data are absent for long chain chlorinated paraffins for ecotoxicity endpoints, data from sources using medium chain chlorinated paraffins will be used to inform the hazard for these endpoints.

 Acute Fish Toxicity
(1)  Johnson and Finely 1980
A series of 96-hour acute fish toxicity studies were conducted by the United States Geological Survey's Columbia National Fisheries Research Laboratory, over the years 1965  -  1978, with several long-chain chlorinated paraffins (Chlorowax LV, C>17, 39% Cl; Chlorowax 40, C>20, 40 - 42% Cl; Chlorowax 50, C>20, 48 - 54% Cl; Chlorowax 70, C>20, 70% Cl) published by Johnson and Finely (1980). Bluegill sunfish (Lepomis macrochirus) and rainbow trout (Oncorhynchus mykiss) were exposed to the test substance (100% commercial formulation) in a static test system. Stock solutions were prepared immediately before each test with acetone used as a carrier solvent. The average pH level was between 7.2 and 7.5 for all tests and the test temperature was 20 +- 1°C for Bluegill sunfish and 10 +- 1 ºC for Rainbow trout. Dilution water hardness ranged from 40 to 50 mg/L as CaCO3. Reported effect levels are considered to be nominal with LC50 values of >300 mg/L for bluegill sunfish and rainbow trout for all LCCPs tested; all values are above the limit of solubility.
                  EPA/OPPT Conclusions
                  Using a weight-of-evidence approach, these studies were considered acceptable to characterize the acute fish toxicity endpoint.
                  96-hr LC50 = NES (> 300 mg/L)

(2) Bengtsson et al. 1979
A 96-hour acute fish toxicity study as part of a bioaccumulation study was published by Bengtsson et al. (1979).  Bleak (Alburnus alburnus) were exposed to a long-chain chlorinated paraffin, Witaclor 549 (C18-26, 49% Cl). The test was performed at 10 °C under semi-static testing conditions. A nominal concentration of 0.125 mg/L was prepared by first dissolving Witaclor in acetone and then added to the dilution water. The treatment vessels and acetone control vessels consisted of six groups of 15 fish (average 4.5 g per fish). The acetone in all treatments did not exceed 0.1 ml/L. The test solutions were renewed every two to three days over the 14-day exposure period. Specific details of the test conditions were not provided other than the tests were performed at 10°C in seawater with a salinity of 7 ppt. Even though the test duration was 14-days, no mortality occurred within 96-hours or 14-days, thus the 96-hour LC50 was > 0.125 mg/L.

                  EPA/OPPT Conclusions
                  This study was considered supplemental to characterize the acute saltwater fish toxicity endpoint using a weight-of-evidence approach.
                   96-hr LC50 = NES (> 0.125 mg/L)

 Acute Aquatic Invertebrate Toxicity
(1) Frank (1993) and Frank and Steinhäuser (1994)
The following study summary (Frank, 1993; Frank and  Steinhauser, 1994), provided in the ECB (2005) MCCP risk assessment, was considered supportive of the aquatic invertebrate hazard determination. The chlorinated paraffin used in these studies was a commercial C14-17 product with a 52 wt% Cl. Daphnia magna were exposed to nominal concentrations of either 100 mg/L or 10,000 mg/L. The 100 mg/L solution was sonicated for 1 hour and then left to stand in the dark for 48 hours before use. The 10,000 mg/L solution also stood for 48 hours in the dark before use, but this time without sonication. After this period, both solutions were filtered firstly with glass filters and then with membrane filters to remove undissolved test substance. The concentrations of medium-chain chlorinated paraffin in the water soluble fractions were then determined by AOX (adsorbable organic halogen) analysis (detection limit of 10 μg/L Cl was equivalent to around 20 μg/L of the chlorinated paraffin). This analysis showed that the concentration of chlorinated paraffin present in the water soluble fraction was around 0.404 - 0.500 mg/L for the 10,000 mg/L nominal solution and 0.071 - 0.142 mg/L for the 100 mg/L stock solution. The acute (48-hour) toxicity tests were carried out using dilutions of the two prepared water soluble fractions. The method used was DIN 38 412, Teil 11, which is equivalent to OECD 202.

In the tests using the water soluble fraction from the 100 mg/L nominal solutions no toxicity was seen at concentrations up to the undiluted stock solution (i.e., no effects up to around 0.071-0.142 mg/L). In experiments using the water soluble fraction from the 10,000 mg/L stock solution, an EC0 of 0.140 mg/L (also reported as 0.100 - 0.110 mg/L in the paper) and an EC25 of 0.423 mg/L (also reported as 0.420-0.470 mg/L in the paper) was determined (maximum mortality seen was 25%) (Frank, 1993). The latter results for the 10,000 mg/L stock solution were reported by Frank and Steinhauser (1994) as EC0 = 0.140 mg/L and EC25 = 0.339 mg/L, and it was noted that some of the Daphnia were floating on the surface of the test solution. In the later study (Frank and  Steinhauser, 1994), the results of further acute toxicity studies were reported using the same test method. An EC50 of 0.037 mg/L and an EC0 of 0.009 mg/L were determined using the water soluble fraction from the 100 mg/L stock solution and no toxic effects were seen in tests with the water soluble fraction from the 10,000 mg/L stock solution (approximately EC0 >= 0.525 mg/L). The authors noted that the effects seen in the acute tests showed poor reproducibility, probably because effects were seen only around the water solubility limit of the substance. However, the authors thought that the possibility of undissolved droplets affecting the results could be ruled out, as floating Daphnia were only sporadically observed in the test.

                  EPA/OPPT Conclusions
                  This study could not be adequately assessed due to inconsistencies in the hazard data for the different dilutions of the test material. In addition, the authors noted that the effects seen showed poor reproducibility, most likely due to effects observed only around the solubility limit in the test system used. The results of this test should therefore be treated with caution, as the effects were mainly seen in the saturated solutions only.

(2)  Tarkpea et al. 1981
The Tarkpea et al. (1981); as quoted in IPCS (1996) summary, provided in the ECB (2005) MCCP risk assessment, was considered supportive of the aquatic invertebrates hazard determination.

The results of tests with the brackish water harpacticoid Nitocra spinipes have been reported (Tarkpea et al., 1981). No other details of the test were reported but the test method was probably the same as reported by Tarkpea et al. (1981), where a static method was employed using water of salinity 7%. at a temperature of 20 - 22°C without aeration, probably using acetone as co-solvent.

                  EPA/OPPT Conclusions
                  This study could not be adequately assessed due to the lack of details provided regarding specific conditions of the test and the preparation of the test solutions. The lack of detail prohibits a review and adequacy of the study. However, the harpaticoid, Nitocra spinipes, is not a standard test species and the test concentrations greatly exceed the limit of solubility. The 96-hour LC50 values for both chlorinated paraffins were identified, but the reliability of the results cannot be determined. Overall, EPA/OPPT reserves judgment on the acceptability of this study. Therefore, data from sources using medium chain chlorinated paraffins will be used to inform the hazard for these endpoints.

 Aquatic Plant Toxicity
(1)  Koh and Thiemann 2001
A 72-hour algae toxicity study using Scenedesmus subspicatus, was conducted at the University of Bremen, Department of Physical and Environmental Chemistry with CP 30 (C17-24, 35% chlorination), CP 40 (C17-20, 44% chlorination) and Hordaflex LC50 (C17-20, 52% chlorination) according to DIN 38412 by Koh and Thiemann (2001). Additional communications with the study author Wolfram Thiemann clarified that nominal test concentrations and local tap water (Bremen, Germany) was used without adjustments however, the study methods were not fully characterized. The stock solutions were prepared to a concentration 6 mg/mL of test substance in acetone. The individual stock solutions were diluted with distilled water to prepare individual standard solutions of 0.250 mg/L for CP 30 and CP 40 and 0.125 mg/L for Hordaflex LC 50. pH was between 5 and 6 and water hardness was between 35.7 and 53.5 mg CaCO3/L. Ambient laboratory air temperature was ~ 21 ºC. The solvent acetone was used to maintain test substance in solution. Effects were calculated based on growth rate; no effects were observed up to 0.250 and 0.125 mg/L.

                  EPA/OPPT Conclusions
                  All three of the chlorinated paraffins tested contain C17 and C18 constituents which are considered to have LCCP-like properties. More information concerning composition would be needed to accept these results for long-chain chlorinated paraffins. Overall, EPA/OPPT reserves judgment on the acceptability of this study.
                  72-hr EC50 (growth rate) = NES (> 0.250 mg/L; CP 30 and CP 40)
                  72-hr EC50 (growth rate) = NES (> 0.125 mg/L; Hordaflex LC 50)
                  72-hr NOEC >= 0.250 mg/L (CP 30 and CP 40)
                  72-hr NOEC >= 0.125 mg/L (Hordaflex LC 50)

 Chronic Fish Toxicity
No data are available.

 Chronic Aquatic Invertebrate Toxicity
(1) Frank 1993 and Frank Steinhäuser 1994
The following data review of Frank (1993) and Frank and Steinhäuser (1994) were directly excerpted from the U.K. Environmental Risk Assessment of long-chain chlorinated paraffins:

   The test substance was identified as C18-20 chlorinated paraffin with 52% chlorination. Frank (1993) carried out a series of acute and longer-term studies with Daphnia magna using a commercial C18-20, 52% wt. Cl product. The tests were carried out using dilutions of the water-soluble fraction of the chlorinated paraffin. Stock solutions of the chlorinated paraffin were made up in water to give nominal concentrations of either 100 mg/L or 10 g/L. The 100 mg/L solution was sonicated for one hour and then left to stand in the dark for 48 hours before use. The 10 g/L solution also stood for 48 hours in the dark before use, but this time without sonication. After this period, both solutions were filtered firstly with glass filters and then with membrane filters to remove undissolved test material (microscopic and spectroscopic investigation of the filtered solutions gave no indication of the presence of droplets) to give the respective water-soluble fractions. The concentration of the chlorinated paraffin in the water-soluble fractions was determined by AOX (adsorbable organic halogen) analysis. The detection limit of the method used was around 10 μg Cl/L, which is equivalent to around 20 μg/L of the chlorinated paraffin. This analysis showed that the concentration of chlorinated paraffin present in the water-soluble fraction was around 462 - 519 μg/L for the 10 g/L nominal solution but was not detectable in the 100 mg/L solution (i.e. <20 μg/L). Experiments were carried out to show that in the test vessels, although the concentration of chlorinated paraffin present fell over time, it remained within 80 per cent of the initial concentration over 2 - 3 days. This time period was used in the long term tests as the renewal period for the solution (semi-static method).

   Long-term (21-day) reproduction studies were also performed using dilutions of the water-soluble fractions of the two stock solutions. The dilutions used were 1:2, 1:4, 1:8 and 1:16 for the 100 mg/L loading and 1:4, 1:8, 1:16, 1:32 and 1:64 for the 10 g/L loading. In these experiments, the test medium was changed three times per week and 10 animals were used per concentration. The tests were carried out at 20°C and a pH of 7.79 - 8.44. Two endpoints were determined in the study: effects on parent mortality and effects on reproduction (number of offspring per adult). Parent mortality in the controls was 0 per cent in the test carried out with the 10 g/L nominal stock solution and 10 per cent in the test carried out with the 100 mg/L nominal stock solution.

   Elevated mortality was seen in the exposed populations. For the 10 g/L stock solution the LOEC was determined as the 1:8 dilution (approximately 58 - 65 μg/L) and the NOEC was determined as the 1:16 dilution (approximately 29 - 32 μg/L). For the 100 mg/L nominal stock solution the LOEC was determined as the 1:4 dilution and the NOEC was determined as the 1:8 dilution. These dilutions are based on the detection limit for the analysis of the 100 mg/L stock solution, and equate to LOEC and NOEC [values] of <5 and <2.5 μg/L respectively). From the dose response curves it appears that 100% parent mortality occurred at a concentrations of around <10 μg/L in the 100 mg/L nominal stock solution experiments and around 125 μg/L in the 10 g/L nominal stock solution experiments.

   For the reproduction endpoint, the average number of young per adult in controls was 72.3 in the 100 mg/L nominal stock solution series of experiments and 73.5 in the 10 g/L nominal stock solution experiments. A significant reduction in the number of young per adult was seen in some of the exposed organisms. For the 100 mg/L nominal stock solution, this effect on reproduction was significantly different from the control groups at the lowest concentration tested (a 1:16 dilution which is equivalent to a chlorinated paraffin concentration of <1.2 μg/L, based on the detection limit of the analytical method used). Thus the NOEC/LOEC for this series of experiments was <1.2 μg/L. Similarly, for the 10 g/L nominal stock solution effects were again seen at the lowest concentration tested (a 1:64 dilution, which is equivalent to a chlorinated paraffin concentration of 7.3 - 8.1 μg/L). This value is treated as the LOEC for this series of experiments. The report also indicates that the NOEC is very close to this value, since using a different statistical method (Dunnett's Test rather than Williams' Test), the effects seen at this concentration were not statistically significantly different from controls.

                  EPA/OPPT Conclusions
                  The interpretation of the results is complicated by the difficulties interpreting the effects from the different loading and dilution concentrations used. The actual exposure concentration in the 100 mg/L nominal stock solution is unknown and the measured concentration in the 10 g/L nominal stock solution (500 ug/L) is above the reported (estimated) water solubility of LCCPs. It was also noted in the U.K. environmental risk assessment, that the data for the 10 g/L loading were reanalyzed by Thompson (2001). The reanalysis suggests that the statistical significance of parent mortality is questionable and that the 1:8 dilution (20% mortality) considered as the LOEC is a marginal effect at best. The NOEC for parent mortality could be considered to be the 1:8 dilution (0.058  -  0.065 mg/L). In addition, there was a serious error found by Thompson (2001) in the statistical method. They determined that the statistical software misinterpreted increasing dilutions as increasing concentrations based on how the data were entered. Re-analysis of the data showed that effects were statistically significant compared with the controls only at the 1:8 dilution, leading to a NOEC at the 1:16 dilution (0.029  -  0.032 mg/L). This problem with the statistical analysis may also explain why the NOEC and LOEC from the 100 mg/L stock were observed below the lowest concentration tested; thus, the NOEC/LOEC of < 0.0012 mg/L is questionable. The data from these studies should be approached with caution due to deficiencies and uncertainties with the statistical analysis. The NOEC and LOEC values that follow are questionable.
                  21-day NOEC = 0.029  -  0.032 mg/L (10 g/L solution; parent mortality)
                  21-day LOEC = 0.058  -  0.065 mg/L (10 g/L solution; parent mortality)
                  21-day NOEC < 0.0025 mg/L (below detection limit; 100 mg/L solution; parent mortality)
                  21-day LOEC <= 0.0050 mg/L (below detection limit; 100 mg/L solution; parent mortality)
                  21-day NOEC < 0.0073 mg/L (10 mg/L solution; reproduction)
                  21-day LOEC <= 0.0073 mg/L (10 mg/L solution; reproduction)
                  21-day NOEC < 0.0012 mg/L (100 mg/L solution; reproduction)
                  21-day LOEC <= 0.0012 mg/L (100 mg/L solution; reproduction)

(2) TNO 1993
The test was carried out according to the OECD [211] methodology using a semi-static test procedure (test solution renewal was carried out every 48 - 72 hours). The test substance was identified as C18-20 chlorinated paraffin with 52% chlorination (Chloroparaffin Hoechst 56 Flüssig, Chloroparaffin Hoechst 52 Flüssig, and Hordaflex LC50. The dilution water used was a synthetic medium (DSWL) prepared by the addition of various salts to ground water. The hardness of the medium was 214 mg/L as CaCO3. The test was carried out using saturated solutions of the chlorinated paraffin using a column technique. The column was prepared by firstly dissolving/suspending 0.1 g of the test substance in 25 mL of acetone. This solution was then added to 10 g of the packing material for the column (chromosorb 60/80 mesh) and the acetone removed by rotation evaporation. The coated packing material was stored at room temperature in the dark until needed. The columns were stainless steel (25 cm long with an internal diameter of 4.3 mm) filled with 1 g of the coated packing material. The column was conditioned by pumping dilution water through at a flow rate of 6.2 mL every three minutes; the first 500 mL was collected and discarded. Around 18 litres of dilution water was then collected in a bottle and continually re-circulated through the column at a flow rate of 3.4 mL per minute throughout the test. The required amount of the saturated solution needed for the start of the test, and at each renewal period, was then taken from the bottle.

Four replicates (10 daphnids in 400 mL of test solution) were carried out for each treatment. The tests were performed in 600 mL beakers and these were conditioned to the test solutions for two days prior to the start of the test. The solutions were renewed every Monday, Wednesday and Friday during the test.

The concentration of test substance was determined at each renewal time in both the "fresh" solution and the "spent" solution. The analytical method used was based on extractable organic halogen (EOX; similar in principle to AOX) analysis. The mean EOX measured in the test solution over the course of the test was around 1 μg/L (the range found in the "fresh" solutions was 1.0 - 1.5 μg/L and the range in the "spent" solutions was 0.5 - 1.5 μg/L. The EOX concentrations in the control solutions were generally <0.5 μg/L in the "fresh" solution but the range found in the spent solutions was <0.5 - 1.0 μg/L for the blank control and 0.5 2.0 μg/L for the column control.

The temperature, DO, and pH during the test were 19.2 - 20.3 ºC, >=7.1 mg/L and 7.6 - 8.5, respectively. The parent survival in both of the control groups was 97.5%. In the C18 - 20, 52% wt. Cl treatment group the parent survival was 92.5%. This survival was not significantly different (at the p=0.05 level) from the control group. Therefore, it was concluded that no treatment related effects on parent morality were seen in the study. The reproduction rate (expressed as the cumulative number of young per living female) in the study was 113.6 in the blank control and 127.3 in the column control. The response of the two controls was not significantly different (at the p=0.05 level). The reproduction rate in the C18 - 20, 52% wt. Cl treatment group was 100.8. This was 88.7 per cent of the blank control response and 79.1 per cent of the column control response. These responses were analyzed statistically in TNO (1993) using the two-tailed Dunnett-test. No statistically significant differences were found between the C18 - 20, 52% wt. Cl treatment group and the blank control, or the column control.

                  EPA/OPPT Conclusions
                  Thompson (2007) conducted a further analysis of the results, due to the author's perception of discrepancies in the UK's (2009) Environmental Risk Assessment. The discrepancy was based on a seemingly contradictory statement from pg. 7 of the TNO report where the reproduction rate of the Hodaflex LC50 and Hoechst 52 Flüssig was stated as being significantly different from the controls (Pg.7 of the report) but not significantly different from the controls (Pg. 16 of the report). The data were re-analyzed by Thompson (2007) and no differences in the reproduction rate were observed between the C18-20, 52 wt% chlorination treatment groups Hodaflex LC50 and Hoechst 52 Flüssig, the column control, blank control or the pooled control group (combined blank control and column control). An additional statistical analysis was conducted by OPPT and no differences were determined for reproduction rate in the treatment groups versus controls using SAS (v. 9.3). Regardless of the discrepancy in the TNO report, statistically significant differences were reported by Thompson (2007) for adult mortality, reproduction rate, and mortality in general for Daphnia exposed to Hoechst 52 Flüssig compared to both controls. However, it is still unclear as to the levels of saturation of each solution the organisms were exposed to based on the extraction technique and nominal loading rates. Therefore, EPA/OPPT will reserve judgment on the acceptability of this study based on a weight of evidence approach.
                  21-day NOEC = 0.002 mg/L (reproduction Hoechst 56 Flüssig)

 Chronic Aquatic Sediment Invertebrate Toxicity
No data are available. Data from secondary sources using medium-chain chlorinated paraffins for chronic aquatic sediment invertebrate toxicity can be used to fill data gaps for the long-chain chlorinated paraffin; and these data were described above in the MCCP section.

 Avian Toxicity
No data are available. Data from secondary sources using medium-chain chlorinated paraffins for avian toxicity can be used to fill data gaps for the long-chain chlorinated paraffins; and these data were described above in the MCCP section.

 Terrestrial Invertebrate Toxicity
No data are available. Data from secondary sources using medium-chain chlorinated paraffins for terrestrial invertebrate toxicity can be used to fill data gaps for the long-chain chlorinated paraffins; and these data were described above in the MCCP section.

 Terrestrial Plant Toxicity
No data are available. Data from secondary sources using medium-chain chlorinated paraffins for terrestrial plant toxicity can be used to fill data gaps for the long-chain chlorinated paraffins; and these data were described above in the MCCP section.

 Conclusions
Sufficient data were available to characterize the acute fish, the acute aquatic invertebrate, the chronic aquatic invertebrate, the chronic aquatic sediment invertebrate, avian, and terrestrial plant toxicity endpoints for MCCPs and LCCPs by read across in some instances. Data for other toxicity endpoints (i.e., chronic fish, aquatic plant, etc.) were inconclusive due to lack of study details, uncertainties in analytical methods, or test material preparation methods; thus, these data are included in order to characterize risk in a qualitative manner, but are used as supportive for the categories under which they are provided. Supporting data were included in order to provide a weight-of-evidence approach used to characterize some endpoints.

Most of the data provided in this review indicated several difficulties were encountered when testing in an aquatic environment. These included: (1) getting the material into solution, (2) measuring the material in solution, and (3) characterizing the effects for each study listed. Often there were many details of a given study omitted, prohibiting a full and robust review of the data. The (estimated) physical-chemical properties of LCCPs and MCCPs (water solubility values between 2 and 30 μg/L and Log Kow values between 4-8) suggest these materials may not partition to the aquatic media or elicit toxicity to aquatic organisms within the water column.

Specifically for the chronic and acute aquatic invertebrate, aquatic sediment, avian, and terrestrial plant endpoints for LCCPs, other analog data provided was acceptable using compounds with chlorination percentage of 52 wt% and carbon chain lengths of C14-17 which is defined as a MCCP material. These data are used in this assessment to fill data gaps for the C18-20 LCCPs as this would be a conservative approach to charactering hazard in the absence of data. Concern concentrations based on these data are again a very conservative approach in the absence of data for the LCCP materials themselves and therefore may not inherently characterize toxicity to LCCPs directly.

The most reliable and acceptable studies indicate that for MCCPs, the toxicity to aquatic organisms for acute endpoints are from the Thompson et al. 1996 study for aquatic invertebrates. Where the 48-hour EC50 value = 0.0059 mg/L. Using the methods described in the Sustainable Futures/P2 Manual (US EPA, 2012), the acute and chronic concentrations of concern (CoC) are derived as follows:

 Acute CoC: The 48-hour EC50 value = 0.0059 mg/L is divided by an assessment factor of 5 to yield an acute concentration of concern (CoC) of 0.00118 mg/L, or 0.001 mg/L, or 1 μg/L (1 ppb). Aquatic Acute Concern Concentration= 1 ppb
 Chronic CoC: The aquatic invertebrate chronic value of 0.013 mg/L from the 1997 Thompson et al. study based on a MCCP material is divided by an assessment factor of 10 to yield 0.0013 mg/L or 1.3 μg/L or 1.3 ppb. Aquatic Chronic Concern Concentration = 1 ppb

For LCCPs, the acute concern concentration may be derived from the Johnson and Finely (1980) studies. For the chronic concern concentration, the results from the Thompson et al. 1997 study based on a MCCP material will be used as a conservative qualitative assessment due to the lack of overly reliable data for this endpoint for LCCPs.

 Acute CoC: Aquatic Acute Concern Concentration= NES
 Chronic CoC: The aquatic invertebrate chronic value of 0.013 mg/L from the 1997 Thompson et al. study based on a MCCP material is divided by an assessment factor of 10 to yield 0.0013 mg/L or 1.3 μg/L or 1.3 ppb. Aquatic Chronic Concern Concentration = 1 ppb (MCCP and LCCP)

The most reliable and acceptable value for the toxicity to aquatic sediment invertebrate organisms acute endpoint is based on the MCCP material from the Thompson et al. 2002 28-d study. For both MCCPs and LCCPs, the 28-d sediment invertebrate GMATC value of 187 mg/kg dry wt sediment is used to assess hazard. The 28-d sediment invertebrate GMATC value of 187 mg/kg dry wt sediment is used to assess hazard. Again, using methods in US EPA (2012):

 Acute CoC: Calculating an acute concern concentration from the chronic value of 187 mg/kg dry wt. The 187 value is multiplied by an acute to chronic ratio for invertebrates (10) to yield 1,870 mg/kg dry wt. This value is then divided by an assessment factor of 5 to yield 374 mg/kg dry wt. Aquatic Sediment Acute Concern Concentration = 374 mg/kg dry wt sediment. (MCCP and LCCP)
 Chronic CoC: The 28-d sediment invertebrate GMATC of 187 mg/kg dry wt sediment is divided by an assessment factor of 10 to yield 18.7 mg/kg dry wt sediment. Aquatic Sediment Chronic Concern Concentration = 19 mg/kg dry wt sediment. (MCCP and LCCP)

The most reliable and acceptable value for the toxicity to terrestrial invertebrates acute endpoint is based on the MCCP material from the Thompson et al. 2001a study. For LCCPs, the 28-d terrestrial invertebrate GMATC value of 149 mg/kg dry wt sediment will be used as an analog approach to assess hazard. The 28-d terrestrial invertebrate GMATC value of 149 mg/kg dry wt sediment from this study will be used. Again, using methods in US EPA (2012):

 Acute CoC: Calculating an acute concern concentration from the chronic value of149 mg/kg dry wt, this value is multiplied by an acute to chronic ratio for invertebrates (10) to yield 1,490 mg/kg dry wt. This value is then divided by an assessment factor of 5 to yield 298 mg/kg dry wt. Terrestrial Invertebrate Acute Concern Concentration = 298 mg/kg dry wt sediment. (MCCP and LCCP)
 Chronic CoC: The 28-d terrestrial invertebrate GMATC of 149 mg/kg dry wt is divided by an assessment factor of 10 to yield 14.9 mg/kg dry wt sediment. Terrestrial Invertebrate Chronic Concern Concentration = 15 mg/kg dry wt sediment. (MCCP and LCCP)

The most reliable and acceptable value for the toxicity to terrestrial plants is based on the MCCP material from the Thompson et al. 2001ab study. For LCCPs, the analog approach using the values from this study may be used. However, there is no OPPT guidance regarding assessing concern concentrations for terrestrial plants.

 HUMAN HEALTH HAZARD STUDY SUMMARIES

 MCCP HEALTH DATA REVIEW

 Metabolism
There is no information on inhalation absorption of MCCPs in humans or in animals. Based on their low vapor pressure and low water solubility, absorption following inhalation or dermal exposure is expected to be limited. An in vitro study using human skin showed that after 24 hours, approximately 0.7% of a C15 chlorinated paraffin was absorbed (Scott, 1984; cited in: ECB, 2008). Oral studies (IRDC, 1984, CXR, 2005; cited in: ECB, 2008) showed that approximately 50% of a single dose of [8-[14]C]-labeled C15 chlorinated paraffin (52 wt% Cl) was absorbed from the GI tract in rats. Excretion via feces was the major route of elimination of radiolabeled material. Elimination of radioactivity from body tissues occurred with an elimination half-life of approximately 2-5 days (liver and kidney) or approximately 2 weeks (adipose tissue).

 Acute Toxicity
There is no information on the effects of a single exposure to MCCPs in humans. No deaths and only limited, non-specific clinical signs of toxicity resulting from exposure of rats to very high doses were observed in an acute oral toxicity study of MCCPs (C14-17, 51-60 wt% Cl); the LD50 was reported to be > 4,000 mg/kg bw (Birtley et al., 1980; cited in: IPCS, 1996). Though no acute toxicity data are available for MCCP by the inhalation or dermal route of exposure, the low acute toxicity data for SCCPs by these routes suggest that MCCPs are likely to have low acute inhalation and dermal toxicity.

 Irritation and Sensitization
No signs of skin irritation were seen with MCCPs (C14-17, 45 wt% Cl), and only slight erythema on the shaved skin was reported in one rabbit at 24 hours exposed to MCCPs (C14-17, 40 wt% Cl) (Chater, 1978; cited in: ECB, 2008). A mild skin irritancy response was reported in one of nine unpublished skin irritation studies of MCCPs (C14-17, 51-60% Cl) in rats (Birtley et al., 1980; cited in: ECB, 2008). The material caused slight, transient eye irritation in rabbits (Birtley et al., 1980; Kuhnert et al., 1986; cited in: ECB, 2008).

No skin sensitization reactions were produced in guinea pig maximization tests conducted on MCCPs (C14-17, 40-45 wt% Cl) (Murmann, 1988; Chater, 1978; cited in: ECB, 2008).

 Repeated-dose Toxicity
There are a number of repeated dose toxicity studies (up to 90-days duration) of MCCPs (C14-17 40 wt% Cl or 52 wt% Cl) in rats by oral exposure (CXR, 2005; Poon et al., 1995; IRDC, 1984; Birtley et al., 1980; and Wyatt et al., 1997; cited in: ECB, 2008). Though the quality and reliability of these studies differs, the liver, kidney, and thyroid were consistently established as the target organs. A summary of the results from these studies is provided in Table 1.

MCCPs caused an increase in liver weight in male rats at exposure levels of > 100 mg/kg-bw/day) and in female rats at exposure levels of > 32 mg/kg-bw/day. Liver enzyme induction was reported in male and female rats starting from 222 and 100 mg/kg-bw/day, respectively. Liver hypertrophy of trace to minimal severity was reported in male rats at dose levels of > 100 mg/kg-bw/day and higher. Collectively, these changes are likely to be related to an increase in metabolic demand as an adaptive response and to peroxisome proliferation, both of which are considered of no or limited toxicological significance to humans. Though Poon et al. (1995) reported various histopathological effects in the liver of male and female rats at dose levels > 36 mg/kg-bw/day, there are a number of deficiencies with this study, including the scoring and classification of histopathological findings and limited reporting of data, which preclude its utility in hazard evaluation. This conclusion is consistent with previous evaluations of this study (ECB, 2008). Further, despite the consistency of findings reported in the review article by Birtley et al. (1980) with other 90-day studies, these findings should be viewed cautiously because the original full study report is not available. Based on the available data, the studies by IRDC (1984) and CXR (2005) provide the most reliable data for identifying effect levels of MCCPs on the liver. For the purposes of this assessment, a NOAEL of 100 mg/kg-bw/day was chosen based on increases in absolute liver weight (i.e., 22-26%), liver hypertrophy of trace severity, and enzyme induction (i.e., 30% increase).

Kidney effects have been reported in a number of studies, with effect levels typically being observed at the limit doses. MCCPs (C14-17 52 wt% Cl) caused significant increases (9-13%) in kidney weight at 222 mg/kg-bw/day (CXR, 2005; cited in: ECB, 2008), as well as "chronic nephritis" and tubular pigmentation in the kidney of female rats at 625 mg/kg-bw/day (IRDC, 1984, cited in: ECB, 2008). One study reported a dose-related increase in congestion starting at 32 mg/kg-bw/day; however, no information was provided on the incidence or severity of this effect (Birtley et al., 1980; cited in: ECB, 2008). An additional study reported minimal to mild hyaline-droplet like cytoplasmic inclusions, starting at > 0.4 mg/kg-bw/day in male rats. This effect is considered of limited relevance to humans. The authors also reported minimal dose-related increases with inner medullary tubular dilation at an incidence of 1/10, 4/10, and 8/10 female rats at 4, 42, and 420 mg/kg-bw/day, respectively (Poon et al., 1995; cited in: ECB, 2008). Though this effect is considered relevant to humans, the study suffers from a number of limitations, which preclude utilizing it for hazard evaluation. However, based on the incidence reported by the authors, the NOAEL of 42 mg/kg-bw/day for kidney effects is consistent with the NOAEL of 23 mg/kg-bw/day reported in the CXR (2005) study. Therefore, a NOAEL of 23 mg/kg-bw/day was chosen for the kidney, based on increases in organ weight at the next highest dose level.

MCCPs (C14-17 52 wt% Cl) have been reported to cause minimal to mild adaptive histopathological changes in the thyroid (i.e., follicular cell hypertrophy and hyperplasia) in two studies in rats starting at 50 ppm (4 mg/kg-bw/day) and above (Poon et al., 1995; IRDC, 1985). Decreased T4 levels and increased TSH levels in the plasma were also seen at similar dose levels. As noted previously, these results have been drawn into question based on the scoring and classification for histopathology, the limited reporting of data, and the inconsistent findings from other more robust studies (ECB, 2008). Therefore, these studies will not be considered further for hazard identification. IRDC (1985) reported mild to moderate hypertrophy and hyperplasia in male rats at dose levels of > 10 mg/kg-bw/day and higher, whereas changes in absolute organ weights of male and female rats were not observed except at the limit dose of 625 mg/kg-bw/day (IRDC, 1985; cited in: ECB, 2008). The remaining studies that evaluated thyroid hormone levels identified a decrease in plasma free T3 in male rats, but not total T3 or free/total T4, and an increase in TSH in female rats at dose levels of 24.6 or 242 mg/kg-bw/day, respectively (CXR, 2005; cited in: ECB, 2008), or fluctuations in thyroid hormones in male or female rats at doses of > 312 mg/kg-bw/day or higher (Wyatt et al., 1997; cited in: ECB, 2008). There is evidence that the thyroid effects observed are attributable to stimulation of this organ arising from a negative feedback effect arising from plasma T4 depletion following increased excretion of this hormone. This depletion of plasma T4 results from the induction of hepatic UDPG-transferase, increased glucuronidation, and ultimately excretion of T4 following exposure to MCCPs. The pituitary responds to the decreased levels of T4 by releasing more TSH, which in turn leads to increased production of T4 by the thyroid. The continuous stimulation of the thyroid in response to the increased excretion of plasma T4 is predicted to ultimately give rise to hypertrophy and hyperplasia in this organ. Humans, unlike rodents, possess T4-globulin binding protein and are therefore less susceptible to plasma T4 depletion and hence any resultant thyroid stimulation. The thyroid effects observed in rats are not considered to be of relevance to chronic human health at relevant levels of exposure, although these changes may be relevant for assessing potential adverse outcomes during reproduction and development, as discussed under Section C-1-7 in this Appendix.

 Genotoxicity
MCCPs (C14-17 40-52 wt% Cl) are not mutagenic to bacteria. Three in vivo bone marrow studies also show that MCCPs are not clastogenic (cited in: ECB, 2008). Therefore, it may be concluded that MCCPs possess a low potential to cause genotoxic effects.

 Carcinogenicity
There is no information on the carcinogenicity of MCCPs; however, carcinogenicity studies on a SCCP and a vLCCP are available. These studies, along with the genotoxicity data on MCCPs, may be used to inform the carcinogenic potential of MCCPs. When administered by gavage, a SCCP (C12, 60 wt% Cl) caused increased incidences of liver tumors in male and female rats, kidney tumors in male rats, and thyroid tumors in female rats (NTP, 1986a). However, based on mechanistic considerations, these tumors are considered to be of little or no relevance to humans.  This conclusion is consistent with previous carcinogenicity evaluations (ECB, 2008). An increased incidence of malignant lymphoma in male mice was reported at the highest dose of 5,000 mg/kg-bw/day in carcinogenicity studies of a vLCCP (C23, 43 wt% Cl) in male and female rats and mice (NTP, 1986b). Because it is unlikely that MCCPs are genotoxic, and the fact that cancer was only observed at the highest dose, the EU (UK 2009) assumed that there was a threshold for this effect in the mice. In addition, there was no increased incidence of malignant lymphoma observed in the carcinogenicity study on an SCCP discussed above. Further, MCCPs are non-genotoxic. Therefore, it may be concluded that MCCPs are unlikely to pose a carcinogenic hazard to humans.

Table_Apx C-1: Summary of Results from 90-day Studies in Rats Administered MCCPs
                             Strain (sample size)
                        Test substance and dose levels
                                 Target organ
                                 Effect levels
F-344
(10 rats/sex/group)[1]
C14-17, 52 wt% Cl

Dietary intake for ♂: 0, 2.38, 9.34, 23.0, or 222 mg/kg-bw/day.

Dietary intake for ♀: 0, 2.51, 9.70, 24.6, or 242 mg/kg-bw/day.
Liver
♂ at 222 and ♀ at 242 mg/kg-bw/day, 13-31% ↑ in organ weight

♂ at 222 mg/kg-bw/day, minimal centrilobular hypertrophy  in 9/10 animals

♂ at 222 mg/kg-bw/day, 82% ↑ in microsomal T4-UDPGA-glucuronyl transferase activity

♀ at 100, 300, and 300 mg/kg-bw/day, 30, 30, and 252% ↑ in microsomal T4-UDPGA-glucuronyl transferase activity, respectively

Kidney
♂ at 222 and ♀ at 242 mg/kg-bw/day, 9-13% ↓ in organ weight

♂ at > 222 and ♀ at 242 mg/kg-bw/day, no treatment-related histopathology

Thyroid
♂ at 222 mg/kg-bw/day, 17% ↑  in plasma TSH

♂ at 23.0 and 222 mg/kg-bw/day, 26% or 22% ↓ in plasma free T3, respectively, but no effects on total T3 or on free/total T4 at any dose

♀ at > 242 mg/kg-bw/day, no effects on free/total T3 or T4

♀at 24.6 and 242 mg/kg-bw/day, 20 and 39% ↑  in plasma TSH

♂at > 222 and ♀ at 242 mg/kg-bw/day, no treatment-related histopathology
Sprague-Dawley
(10 rats/sex/group)[2]
C14-17, 52 wt% Cl

Dietary intake for ♂: 0, 0.4, 4, 36, or 360 mg/kg-bw/day.

Dietary intake for ♀: 0, 0.4, 4, 42, or 420 mg/kg-bw/day.
Liver
♂ at 360 and ♀ at 420 mg/kg-bw/day, 28 and 48% ↑ in absolute and relative weights, respectively

♂ and ♀ at < 4 mg/kg-bw/day, no treatment-related histopathology

♂at 36 and ♀ at 42  mg/kg-bw/day, minimal increase in anisokaryosis and vesiculation of the nuclei

♂ at 360 and ♀ at 420 mg/kg-bw/day, mild increase in anisokaryosis and vesiculation of the nuclei (7-10 animals)

♂ at 360  mg/kg-bw/day, ↑ in perivenous homogeneity

♀ at 42 and 420 mg/kg-bw/day, ↑ in perivenous homogeneity

♂ at 360  and ♀ at 420 mg/kg-bw/day, ↑ in single cell necrosis (incidence not reported)

Kidney
♂ at 360 and ♀ at 420 mg/kg-bw/day, 11% ↑ in absolute and relative weights

♂ at > 0.4 mg/kg-bw/day, minimal to mild hyaline-droplet like cytoplasmic inclusions, with significant accumulation at the limit dose

♀ at > 4 mg/kg-bw/day, minimal dose-related inner medullary tubular dilation seen in 0/10, 0/10, 1/10, 4/10, and 8/10 animals

Thyroid
♂ at 36 and 360 mg/kg-bw/day, minimal to mild morphological changes affecting the architecture (i.e., reduced follicle sizes and collapsed angularity) and the epithelium (i.e., increased height, cytoplasmic vacuolation, and nuclear vesiculation)

♀ at > 4 mg/kg-bw/day, minimal to mild morphological changes affecting the architecture (i.e., reduced follicle sizes and collapsed angularity) and the epithelium (i.e., increased height, cytoplasmic vacuolation, and nuclear vesiculation)
F-344
(15 rats/sex/group)[3]
C14-17, 52 wt% Cl

Dietary intake for ♂ and ♀: 0, 10, 100, or 625 mg/kg-bw/day.

Liver
♂ and ♀ at 100 and 625 mg/kg-bw/day, 22-26% and 64-92% ↑ in absolute weight values, respectively

♂ at 100 and 625 mg/kg-bw/day, hypertrophy of trace severity seen in 1/15 and 13/15 animals, respectively

♀ at 625 mg/kg-bw/day, hypertrophy of trace severity seen in 13/15 animals

Kidney
♂ and ♀ at 625 mg/kg-bw/day, 18% ↑ in absolute weight values

♂ at > 10 mg/kg-bw/day, trace to mild nephritis seen in 1/15, 3/15, 4/15, and 10/15 animals

♀ at 625 mg/kg-bw/day, tubular pigmentation (9/14 animals)

Thyroid
♂ at 625 mg/kg-bw/day, 50% ↑ in absolute weight values

♂ at > 10 mg/kg-bw/day, mild to moderate hypertrophy observed in controls with a dose-dependent trend towards ↑ severity in treated animals

♂ at > 10 mg/kg-bw/day, trace to mild hyperplasia with a dose-dependent trend towards ↑ severity

Adrenal
♂ and ♀ at 625 mg/kg-bw/day, 25% ↑ in absolute weight values
Wistar-derived
(24 rats/sex/group)[4]
C14-17, 52 wt% Cl, containing epoxidized vegetable oil as a stabilizer

Dietary intake for ♂: 0, 33, 167, or 333 mg/kg-bw/day.

Dietary intake for ♀: 0, 32, 160, or 320 mg/kg-bw/day.

Liver
♂ at 167 and 333 mg/kg-bw/day, 15 and 22% ↑ in relative weight values, respectively

♀ at 32, 160, and 320 mg/kg-bw/day, 11, 21, and 48% ↑ in relative weight values, respectively

♂ at 333 and ♀ at 320 mg/kg-bw/day, no histopathological abnormalities

♂ at > 33 and ♀ at > 32 mg/kg-bw/day, dose-related ↑ in proliferation of smooth endoplasmic reticulum (electron microscopy)

Kidney
♂ and ♀ at limit dose, 15% ↑ in relative weight

♂ and ♀, dose-related ↑ in congestion (incidence and severity not reported)

♂ and ♀ at > limit dose, no histopathological abnormalities
F-344
(10 rats/sex/group)[5]
C14-17, 40 wt% Cl

Oral gavage for ♂ and ♀: 0, 312, or 625 mg/kg-bw/day
Liver
♂ and ♀ at 312 and 625 mg/kg-bw/day, 37 and 72% ↑ in relative weight, respectively, (absolute weight and bodyweight not presented)

♂ and ♀, dose-related ↑ in centrilobular hypertrophy (incidence and severity not reported)

♂ and ♀ at 312 and 625 mg/kg-bw/day, dose-related ↑ in β-oxidation from day 29 onwards (~2.7- and 3.3-fold ↑, respectively, at study termination)

♂ and ♀ at 312 and 625 mg/kg-bw/day, dose-related ↑ in UDPG-transferase activity from day 15 onwards (up to 100% ↑, respectively)

Thyroid
♂ and ♀ at 312 and 625 mg/kg-bw/day, ↓ in levels of free and plasma T3, which reached statistical significance on days 15 and 57

♂ at 312 and 625 mg/kg-bw/day, ↑ TSH up to 2-fold on day 8 only

♀ at 312 and 625 mg/kg-bw/day, T3 significantly ↑ by day 91

♀ at 312 and 625 mg/kg-bw/day, total plasma T4 significantly ↓ by up to 25% on day 57

♂ and ♀ at 312 and 625 mg/kg-bw/day, ↑ follicular cell hypertrophy throughout the study, and accompanied by follicular cell hyperplasia on days 55 and 91 (incidence and severity not reported)

♂ and ♀ at 312 and 625 mg/kg-bw/day, significantly ↑ replicative DNA synthesis on day 29, but not on day 91
[1] CXR (2005), cited in: ECB (2008).
[2] Poon et al. (1995), cited in: ECB (2008).
[3] IRDC (1984), cited in: ECB (2008).
[4] Birtley et al. (1980), cited in: ECB (2008); note, this study was only summarized in the review by Birtley et al. (1980). The underlying original study report was not available.
[5] Wyatt et al. (1997), cited in: ECB (2008).

 Developmental Reproductive Toxicity Review
A series of range-finding and definitive prenatal developmental and reproductive toxicity studies were conducted in rats and rabbits with medium-chain chlorinated paraffins (MCCPs). These studies were conducted between 1981 and 1986. They appear to be valid toxicity studies, conducted according to the standard methodologies available at the time. More recently, additional studies with MCCPs have been conducted in an attempt to determine the cause of hemorrhaging in the pups observed in a one-generation reproductive toxicity range-finding study.

In several prenatal developmental toxicity studies with MCCPs conducted via gavage, no signs of maternal toxicity were seen at doses as high as 500 mg/kg-bw/day in rats and 100 mg/kg-bw/day in rabbits. Likewise, no signs of developmental toxicity were observed at doses as high as 5000 mg/kg-bw/day in rats and 100 mg/kg-bw/day in rabbits.

Two reproductive toxicity studies with MCCPs in rats have been conducted. A one-generation reproductive toxicity range-finding study showed that administration of approximately 100 and 400 mg/kg-bw/day MCCPs via the diet had no effect on fertility or other reproductive parameters; however, internal hemorrhaging and deaths in pups were observed beginning from 74 mg/kg-bw/day (1000 ppm) up to approximately 400 mg/kg-bw/day (6250 ppm). These effects in the pups were not seen in a more recent definitive one-generation reproductive toxicity study with exposure to MCCPs for 11-12 weeks to doses as high as 100 mg/kg-bw/day (1200 ppm). Internal hemorrhaging was not seen in the adult animals in either of these studies at doses as high as 400 mg/kg-bw/day (6250 ppm), or in another study in non-pregnant female rats repeatedly exposed to doses as high as 1000 mg/kg-bw/day. However, when dams were exposed to approximately 500 mg/kg-bw/day (6250 ppm) MCCPs during cohabitation, gestation, and lactation, signs of hemorrhaging were observed in dams that died at the time of parturition. Taken together, the results of these studies suggest that newborns during lactation and pregnant females at the time of parturition are a potentially sensitive subpopulation.

The UK Risk Assessment (February, 2008) did not use the LOAEL of 74 mg/kg-bw/day (1000 ppm) from the one-generation reproductive toxicity range-finder study as a point of departure because the pup deaths at that dose were not statistically significant. The study itself used a limited number of animals and was intended for dose range-finding purposes only and, more importantly, the pup deaths were not repeated in a more recently conducted definitive study. With respect to developmental/reproductive toxicity, the UK Risk Assessment identified two subpopulations at risk: offspring during lactation and pregnant dams at parturition. The NOAELs from the definitive one-generation reproductive toxicity study (a maternal NOAEL ~ 47 mg/kg-bw/day (600 ppm) for effects on the offspring mediated via lactation; and a maternal NOAEL ~ 100 mg/kg-bw/day (1200 ppm) for effects on the dam during the time of parturition) were used to calculate risk. Assuming a conservative value of 50% oral absorption, the margin of safety (MOS) for effects on the offspring mediated via lactation and effects on the dam during the time of parturition were calculated for workers, consumers, and other scenarios. In all but one scenario (oil-based metal working fluids), the margins of safety were above 100 and in many cases, several fold above. In addition, margins of exposure were calculated for infants exposed via breast milk and via cow's milk, and in both instances, large MOEs (i.e., > 100) were calculated.

Additional studies with MCCPs have been conducted in an effort to clarify the possible causes of the hemorrhaging in the pups. One (single-dose; 6250 ppm or 538 mg/kg-bw/day) study showed maternal death during parturition due to low levels of vitamin K and related hemorrhaging, suggesting that the act of parturition places dams at higher risk. It was concluded in data from this study and a cross-fostering study that the fetus relies on clotting factors via mother's milk and severe deficiencies in vitamin K levels and related clotting factors in the pups results in hemorrhaging.

No definitive developmental neurotoxicity studies on MCCPs were located. It is not clear if any developmental neurotoxicity endpoints were actually measured in the available prenatal developmental/reproductive toxicity studies; none were explicitly stated. The only information available regarding behavior during development is from cage-side observations in pups through LD 21. In these cases, no dose-related differences were reported in F1 post-weaning appearance or cage-side behaviors.

In the prenatal developmental toxicity study in rats, the LOAEL for maternal toxicity was 2000 mg/kg-bw/day based on clinical signs. The NOAEL for maternal toxicity was 500 mg/kg/day. The NOAEL for developmental toxicity was 5000 mg/kg-bw/day, the highest dose tested.

In the prenatal developmental toxicity study in rabbits, no adverse, treatment-related effects were reported in the dams or the offspring. The NOAEL for both maternal and developmental toxicity was 100 mg/kg-bw/day, the highest dose tested.

In the reproduction range-finding study in rats (IRDC, 1981, cited in ECB, 2008), the LOAEL for maternal toxicity was 6250 ppm (463 mg/kg-bw/day) based on reductions in body weight gains. The NOAEL for maternal toxicity was 1000 ppm (74 mg/kg-bw/day). The LOAEL for developmental toxicity was 1000 ppm (62/74 mg/kg/day) based on pup mortality associated with internal hemorrhages. The NOAEL for developmental toxicity was 100 ppm (6/8 mg/kg-bw/day). No effects on any reproductive parameters were reported. The NOAEL for reproductive toxicity was 6250 ppm (384/463 mg/kg-bw/day).

In the one-generation reproduction toxicity study in rats, the LOAEL for maternal toxicity was 1200 ppm (~100 mg/kg-bw/day) based on increases in liver weight; the NOAEL for maternal toxicity was 600 ppm (~ 47 mg/kg-bw/day). The NOAEL for developmental and reproductive toxicity was 1200 ppm (~ 84/99 mg/kg-bw/day), the highest dose tested.

Basis for Conclusions

In a range-finding prenatal developmental toxicity study in pregnant Charles River COBS CD rats administered MCCPs (C14-17, 52 wt% Cl) via gavage at dose levels of 0, 1000, 1500, and 2500 mg/kg-bw/day on gestation days (GD) 6-20, no effects were observed in the dams at doses up to 2500 mg/kg-bw/day (IRDC, 1983, 1984; cited in: ECB, 2008). As a result, doses greater than 2500 mg/kg-bw/day were selected for the definitive study.

In the definitive study, four groups of 25 pregnant Charles River COBS CD rats were administered MCCPs (C14-17, 52 wt% Cl) via gavage at doses of 0, 500, 2000, and 5000 mg/kg-bw/day on GD 6-19 (IRDC, 1984; cited in: ECB, 2008). Unmated males and females were individually housed and acclimated for 21-days in an environmentally controlled room. At the end of the acclimation period, all animals were weighed and subjected to a detailed physical examination. One female and one male rat were placed together for mating. Confirmation of mating was based on evidence of a copulatory plug or by vaginal smear for sperm. The day mating was confirmed was designated as day 0 of gestation. Test article was administered to pregnant females orally by gavage as a single daily dose on GD 6-19. During treatment, pregnant females were observed daily for mortality and clinical signs of toxicity. Any females not surviving to scheduled sacrifice were necropsied. Body weights were recorded on GD 0, 6, 9, 12, 16, and 20. All females were sacrificed on GD 20 and the uterus and ovaries excised for examination. The number and location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed. The abdominal and thoracic cavities underwent gross examination. Maternal tissues were preserved for future histopathological analysis. Fetuses were weighed, sexed, tagged, and examined for external malformations and variations, including the palate and the eyes. The fetuses underwent visceral and skeletal examinations for malformations and developmental variations.

The only effects reported in dams consisted of an increased incidence in wet matted and yellow stained haircoat in the anogenital area at 5000 mg/kg-bw/day, and soft stool at > 2000 mg/kg-bw/day. No treatment-related adverse effects were reported in offspring at doses up to 5000 mg/kg-bw/day. The LOAEL for maternal toxicity was 2000 mg/kg-bw/day based on clinical signs; the NOAEL for maternal toxicity was 500 mg/kg-bw/day. The NOAEL for developmental toxicity was 5000 mg/kg-bw/day, the highest dose tested.

In a range-finding prenatal developmental toxicity study in pregnant Dutch Belted rabbits administered MCCPs (C14-17, 52 wt% Cl) via gavage at dose levels of 0, 100, 300, 1000, 2000, and 3000 mg/kg/day on GD 6-27, an increase in the number of abortions was observed at > 1000 mg/kg/day (IRDC, 1985; cited in: ECB, 2008). Body weight reductions in the dams were reported at 100 and 300 mg/kg/day. As a result, another range-finding prenatal developmental toxicity study in rabbits was initiated. This second range-finding study showed decreases in maternal weight gain at 80 and 160 mg/kg-bw/day (IRDC, 1982b; cited in: ECB, 2008).

Based on the results of these range-finding studies, dose levels of 10, 30, and 100 mg/kg-bw/day were selected for the definitive prenatal oral gavage developmental toxicity study (IRDC, 1983; cited in: ECB, 2008). In the definitive study, four groups of 16 pregnant Dutch Belted rabbits were administered 0, 10, 30, and 100 mg/kg-bw/day MCCPs (C14-17, 52 wt% Cl) via gavage on GD 6-27. Unmated males and females were individually housed and acclimated for 50-days in an environmentally controlled room. As a result of a positive finding for parasites in stool samples collected during acclimation, all rabbits received sodium sulfamethazine in their drinking water for 16 days during the acclimation period. This treatment was terminated 4 weeks prior to study initiation and only rabbits testing negative for parasites were placed on study. At the end of the acclimation period, all animals were weighed and subjected to a detailed examination. Females were impregnated via artificial insemination. Three weeks prior to artificial insemination, females were given chorionic gonadotropin via an injection in a marginal ear vein in order to induce superovulation. Semen was collected from males of proven fertility and evaluated for motility. The day of artificial insemination was designated as day 0 of gestation. During treatment, pregnant females were observed for mortality and clinical signs of toxicity. Body weights were recorded on GD 0, 6, 12, 18, 24, and 28. Any females not surviving to scheduled sacrifice were necropsied. On GD 28, all surviving females were sacrificed and the uterus and ovaries excised for examination. The location and number of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed. The thoracic and abdominal cavities underwent gross examination. Pooled samples of abdominal adipose tissue from 3 dams were frozen for future analysis. Each fetus was sexed, weighed, and examined for external malformations and variations, including the palate and the eyes, as well as visceral and skeletal examinations for malformations and developmental variations, including examination of the brain and the heart.

No adverse, treatment-related effects were reported in the dams or the offspring at doses up to 100 mg/kg-bw/day. The NOAEL for both maternal and developmental toxicity was 100 mg/kg-bw/day, the highest dose tested.

In a one-generation reproductive toxicity range-finding study, four groups of 5 male and 10 female Charles River COBS SC rats were administered MCCP (C14-17, 52 wt% Cl) via the diet at 0, 100, 1000, and 6250 ppm (~ 0, 6, 62, and 384 mg/kg-bw/day, respectively, in males; and 0, 8, 74, or 463 mg/kg-bw/day, respectively, in females) (IRDC, 1985; cited in: ECB, 2008). F0 animals were exposed to test substance from 28 days prior to mating until sacrifice; F1 animals were treated from weaning until sacrifice, with additional potential exposures occurring in utero and during lactation. All F0 males were sacrificed after the mating period. Following the premating period, each male was cohabited with two females for 10 days. Females were examined for evidence of copulation by means of vaginal smears and/or the appearance of a vaginal plug. The day evidence of copulation was determined was designated as day 0 of gestation. Direct dosing began at 83 days of age for the F0 parents and at 21 days of age for the F1 weanlings. The F0 and F1 animals were observed for clinical signs of toxicity, changes in general appearance and behavior, and mortality. In the F0 adults, body weights and food consumption were measured weekly; in addition, body weights were measured in F0 females on GD 0, 7, 14, and 20; and on lactation days (LD) 0, 7, 14, and 21. Estrous cyclicity was determined in F0 females prior to mating, during mating, and prior to dosing. All F0 females were allowed to deliver. The day the entire litter was found and delivery was judged to be complete was designated as LD 0. Gestation duration was calculated. Following delivery, all pups were examined for external malformations and the numbers of live births and stillbirths (litter size) was recorded for each dam. Pups were weighed, sexed, and examined externally on LD 0, 7, 14, and 21. Litter size was determined on LD 0, 4, 10, and 21. The number of male and female pups was recorded on LD 4. Litters were examined daily for survival. F0 females were examined for behaviors in nesting and nursing. On LD 21, all dams were sacrificed and a gross necropsy performed, including examination of the uterine contents for implantation sites; and ten F1 weanlings/sex/dose were sacrificed and necropsied. Five F1 males and ten F1 females/dose group were retained after LD 21 and sacrificed at 70 days (10 weeks) of age and necropsied. Due to high mortality in high-dose F1 pups, the surviving F1 pups in the high-dose group and an equal number of control pups were sacrificed on LD 6 and 7 and necropsied. Blood was collected via heart puncture and complete blood counts performed. Bone marrow smears were collected from the femur, and the abdominal contents of the pups with milk in the stomach were collected and frozen for future analyses.

Effects in the adults consisted of isolated reductions food consumption and body weight in the dams at 6250 ppm. Effects in the offspring consisted of significant reductions in pup survival at the high dose (none of the F1 pups in the high-dose group survived until lactation day 21); and slight (11%, not statistically significant) decreases in pup survival, and labored breathing, subcutaneous hematoma, pale discoloration, blood around the orifices, pale liver, kidney, and spleen, and blood in the cranial cavity and brain beginning at the mid-dose. No dose-response, treatment-related adverse effects were reported in the offspring in the low dose group. Reductions in body weight in F1 male and female pups occurred during LD 7, 14, and 21, but these reductions were not statistically significantly different from controls, and were seen only in the low- and mid-dose groups but not the high-dose group. There were no dose-related differences in F1 post-weaning appearance, behavior, food consumption, or clinical or anatomical pathology in the low- and mid-dose groups. Based on the results of this study, it was recommended that dosage levels in a two-generation reproduction toxicity study not exceed 1000 ppm. The LOAEL for maternal toxicity was 6250 ppm (~463 mg/kg-bw/day) based on reductions in body weight gains. The NOAEL for maternal toxicity was 1000 ppm (~74 mg/kg-bw/day). The LOAEL for developmental toxicity was 1000 ppm (~62/74 mg/kg-bw/day) based on pup mortality due to hemorrhaging. The NOAEL for developmental toxicity was 100 ppm (~6/8 mg/kg-bw/day). No effects on any of the reproductive parameters were reported. The NOAEL for reproductive toxicity was 6250 ppm (~384/463 mg/kg-bw/day).

In an effort to determine the cause of hemorrhaging in the pups at the high dose from the reproductive toxicity range-finding study, a screening level cross-fostering developmental toxicity study was conducted in Charles River COBS Wistar rats fed diets containing either 0 or 6250 ppm (~ 3125 mg/kg-bw/day) MCCPs (C14-17, 52 wt% Cl) for 4 weeks prior to mating and throughout pregnancy in a series of groups (Hart et al., 1985; cited in: ECB, 2008). Offspring from two of these groups (pups from control females reared from treated females, and pups reared from their treated mothers) showed high-pup mortality associated with internal hemorrhages. Hematological assays in the pups from these two groups showed decreases in factor X, resulting in a disruption of a vitamin K-dependent clotting system (lower plasma vitamin K levels). It was concluded that the pup mortalities were due to internal hemorrhages caused by a decrease in the vitamin K-dependent hemostatic mechanism (not examined in this study), induced during lactational exposures via the milk from mothers receiving MCCPs.

Additional studies have been conducted to investigate two hypotheses in an effort to clarify the possible causes of the hemorrhaging in the pups.

The first hypothesis proposes that MCCPs induce a catabolism of vitamin K in lactating rats leading to decreased plasma concentrations and ultimately low levels of vitamin K in the milk pups receive (vitamin K controls the formation of several clotting factors in the liver). In order to test this hypothesis, a preliminary study (CXR Biosciences Ltd., 2003; cited in: ECB, 2008) was conducted in which three groups of 6 female adult Sprague-Dawley were administered MCCPs (C14-17, 52 wt% Cl) via gavage at doses of 0, 500, or 1000 mg/kg-bw/day for 21 days while being fed a normal diet or a vitamin K-deficient diet. Following exposures to MCCPs, significant decreases in plasma concentrations of a clotting factor were seen in rats fed a normal diet; however, these decreases did not affect prothrombin clotting times. Reductions of a clotting factor in both treated and control groups were also seen in animals fed a vitamin-K deficient diet. Plasma vitamin K levels were not affected by treatment in the normal diets, but they were lower in high-dose animals fed vitamin K-deficient diets. The results from this study suggested that MCCPs did not adversely affect the blood clotting system in adult female rats treated for 3 weeks up to a dose of 1000 mg/kg-bw/day; and the hemorrhaging effects in pups are unlikely to be mediated by reduced vitamin K levels in breast milk.

The second hypothesis proposes that MCCPs transferred to the pups through breast milk causes disruption of the pup clotting system. In order to test this hypothesis, a study (CXR Biosciences Ltd., 2004; cited in: ECB, 2008) was conducted in two groups of 16 male and 32 female Sprague-Dawley rats administered 0 or 6250 ppm (~ 0 and 513 and 538 mg/kg-bw/day in males and females, respectively) MCCPs (C14-17, 52 wt% Cl) for 4 weeks prior to mating, during cohabitation, gestation, and lactation until study termination (at about 2 weeks after the first litters were born, due to high rate of pup mortality). Milk, blood, and liver samples from lactating dams, and blood and liver samples from lactating pups were assessed for plasma vitamin K levels. Five dams died or were killed at the time of parturition (16% mortality). These deaths were considered to be treatment-related as there was no indication of obstruction or hindrance to delivery. The clinical necropsy of these dams showed effects suggestive of hemorrhaging in 3 out of the 5 dams and one male who died. Slight reductions in food consumption and body weight gains were observed during gestation and lactation. There were no effects on mating performance or duration of gestation. Concentrations of plasma vitamin K levels in adult females having gone through lactation and pregnancy was markedly decreased by treatment with MCCPs, which in turn produced a decrease in activity of the plasma clotting factors in treated dams. Prothrombin clotting times were not affected in the dams, suggesting that the functional reserve in these adult animals was sufficient. Pup plasma volumes were reportedly insufficient to measure vitamin K directly, but clotting factor activities were possible to analyze. No effects on litter size at birth or on pup mortality from birth to LD 4 were reported; however, after pup mortality increased significantly after LD 4. The majority of these pups showed internal hemorrhages at necropsy. It was concluded that data from this study and the cross-fostering study performed by Hart et al. (1985) suggest that the fetus receives sufficient vitamin K via the placenta, but after birth becomes severely deficient in vitamin K and related clotting factors and relies on these factors via mother's milk. In addition, the pups also receive considerable levels of MCCPs via lactation (through mother's milk) which may also contribute to further reducing the vitamin K levels. These severe deficiencies in vitamin K levels and related clotting factors in the pups results in hemorrhaging. It was also concluded that the act of parturition places dams at higher risk.

More recently, a definitive one-generation reproductive toxicity study was conducted to refine the NOAEL for effects in the offspring and to further explore the mechanisms of hemorrhaging (CXR, 2006; cited in: ECB, 2008). This study was reportedly conducted in compliance with OECD TG 421 and Good Laboratory Practice standards. Four groups of 12-17 male and female Sprague-Dawley rats were administered 0, 300, 600, and 1200 ppm (~ 0 and 21, 44, and 84 mg/kg-bw/day in males; and 0, 23, 47, and 99 mg/kg-bw/day in  females) MCCPs (C14-17, 52 wt% Cl) for 4 weeks prior to mating, during cohabitation, gestation, and lactation until study termination (for a total treatment of 11-12 weeks). Males were terminated on LD 4 (9 weeks of treatment) and females were allowed to litter and rear their offspring until PND 21. Females were sacrificed on LD 21. Adult males were assessed for signs of clinical toxicity, body weight, food consumption, and macropathology. Adult females were assessed for signs of clinical toxicity, body weight, food consumption, gestation length, parturition, liver weights, and macropathology. Mating performance and fertility were also evaluated. Offspring evaluations included clinical signs of toxicity, litter size, survival, sex ratio, body weight, and pathological examinations at necropsy. Milk, blood, and liver samples were obtained from selected offspring at specific time points between birth of litters and PND 21. In addition, blood, liver, and milk samples from a satellite group of five females and their litters from the control and high-dose group (1200 ppm) were collected for future analysis. Analysis of these samples was still pending at the time of the UK assessment.

No adverse effects were reported in the adult animals for clinical condition, body weight, body weight gain, food consumption, estrous cycling, mating performance, pre-coital interval, fertility, number of implantations, gestation lengths, or parturition. The only effect reported was for higher absolute and relative liver weights in high-dose females (1200 ppm; 99 mg/kg-bw/day). Likewise, no adverse effects were in the offspring at any dose level for litter size, sex ratio, offspring survival, body weights, body weight gains, macropathology and liver weights. No adverse effects were reported on pre- and post-natal survival and growth up to sacrifice (weaning). Though no histopathology was performed, the body cavity and cranial cavity were opened and examined for any signs of hemorrhaging. None was reported. Based on the results of this study, the LOAEL for maternal toxicity was 1200 ppm (~100 mg/kg-bw/day) based on increases in liver weight; the NOAEL for maternal toxicity was 600 ppm (~ 47 mg/kg-bw/day). The NOAEL for developmental and reproductive toxicity was 1200 ppm (~ 84/99 mg/kg-bw/day), the highest dose tested.

 LCCP HEALTH DATA REVIEW

P-12-0433 is a C18-20 LCCP. Much of the information presented here is based on data with vLCCPs (i.e., carbon chain lengths of >C20).

The National Research Council (NRC, 2000) reviewed the toxicological risks of selected flame retardant, including a LCCP containing C24 with 70 wt% Cl. Based on the NOAEL of 900 mg/kg-bw/day (liver toxicity), the NRC derived an RfD of 0.3 mg/kg-bw/day. Using this RfD and the worst case average daily exposure to be 0.16 mg/kg/day, NRC concluded: "LCCP do not pose a noncancer risk when incorporated into residential furniture at the estimated application levels." Further, it was concluded that: "LCCP are not likely to be a human carcinogen and derivation of a cancer potency factor is unnecessary."

 Metabolism
There is no information on inhalation absorption of LCCPs in humans or in animals. Based on their low vapor pressure and low water solubility, absorption following inhalation or dermal exposure is expected to be limited.
Oral (gavage) studies (IRDC, 1981; cited in: UK, 2009) showed that approximately 82-95% of a single dose of [8-[14]C]-labeled C22-26 chlorinated paraffin (43% Cl) was recovered in the feces in rats during the seven-day collection period. Only 0.1-0.8% of the radiolabel was excreted in the urine. For C22-26 chlorinated paraffin (70 wt% Cl), it was found that 61-88% of the administrated radioactivity was recovered in the feces in rats during the seven-day collection period. Less than 0.1-1% was excreted in the urine.

 Acute Toxicity
Acute toxicity studies have been conducted in rats, mice or dogs on five LCCPs: C20-30 , 41-50 wt% Cl; C22-26, 42 wt% Cl, C23, 43 wt% Cl; C20-30, 61-70 wt% Cl; C24, 70% Cl. The maximum dose levels used in these studies ranged from 4-50 g/kg-bw. No deaths were reported in any of the studies (IUCLID, 2003; cited in: UK, 2009).

 Irritation and Sensitization
Skin irritation testing has been conducted on four LCCPs: C19 ,44 wt% Cl; C22-26, 42 wt% Cl; C20-30, 41-50 wt% Cl; C20-30, 70 wt% Cl. No evidence of irritation was seen in three of the four LCCPs. For the C22-26, 42 wt% Cl product, erythema was observed in two of six animals tested; the severity threshold was below the classification of the EU system (IUCLID, 2003; cited in: UK, 2009).

Evidence of slight eye irritation was seen in a test of a C22-26, 42 wt% Cl product. However, the criteria for classification as an eye irritant were not met (IUCLID, 2003; cited in: UK, 2009).

In a maximization test and a Buehler test using guinea pig, a C22-26, 42 wt% Cl product was negative (IUCLID, 2003; Bailey and Sheldon, 1998; cited in: UK, 2009). A C18-27, 40 wt% Cl product was reported to elicit a positive response in the guinea pig maximization test (IUCLID, 2000; cited in: UK, 2009). However, no information is available on the quality of this study.

 Repeated-dose Toxicity
LCCPs with C20-30, 43 wt% Cl were dissolved in corn oil and given by gavage at 100, 900 or 3,750 mg/kg-bw/day to groups of 15 male and female Fisher 344 rats in a 14- and a 90-day studies (IRDC1981, 1984; cited in: Serront et al., 1987). There was a treatment-related effect on the liver of female rats at all dose levels, but no liver effects were seen in the males. Female liver weights were increased and a multifocal granulomatous hepatitis characterized by inflammatory changes and necrosis. Nephrosis was observed in the kidney of male rats and mineralization in the kidneys of female rats at 3,750 mg/kg-bw/day. Similar liver effects were observed in the high-dose (3,750 mg/kg-bw/day) rats of both sexes in a 90-day study on LCCPs with C22-26, 70 wt% Cl (IRDC1981; cited in: Serront et al., 1987) and in a 90-day study as well as a 2-year bioassay on a LCCP with an average of C23, 43 wt% Cl at 100 mg/kg-bw/day (NTP, 1986). Based on the liver toxicity, a LOAEL of 100 mg/kg-bw/day is established for the LCCPs with 43 wt% Cl and a NOAEL of 900 mg/kg-bw/day is identified for the LCCPs with 70 wt% Cl.
 Genotoxicity
Both LCCPs of C22-26, 43 wt% Cl and C23, 43 wt% Cl were negative in several Salmonella strains of the Ames test with or without metabolic activation (IUCLID, 2003; NTP, 1986; cited in: UK, 2009). LCCPs with C20-30 with 43 wt% Cl or C22-26 with 70 wt% Cl did not induce significant increases of chromosomal or chromatid aberrations in bone marrow cells of rats (IRDC1983; cited in: Serrone et al., 1987).

 Carcinogenicity
The carcinogenicity of a vLCCP (C23, 43 wt% Cl) was studied 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 (NTP, 1986). Male rats received doses of 0, 1,875 or 3,750 mg/kg-bw/day; female rats and mice received 0, 2,500 or 5,000 mg/kg-bw/day. An increased incidence of malignant lymphomas was reported for male mice given the LCCP; the incidences for the controls, low- and high dosed groups are 6/50, 12/50, and 16/50, respectively. vLCCPs are not genotoxic (see review in Appendix C, Section C-2-6), and the fact that cancer was only observed at the highest dose, the EU (UK 2009) assumed that there was a threshold for this effect in the mice. In addition, there was no increased incidence of malignant lymphoma observed in the carcinogenicity study on an SCCP (also discussed above in the MCCP section). It has been concluded that LCCPs are unlikely to pose a carcinogenic hazard to humans (NRC, 2000; Serrone et al., 1987).
 Developmental Reproductive Toxicity Review
In a prenatal developmental toxicity study in rats, no treatment-related effects were reported. The NOAEL for both maternal and developmental toxicity was 5000 mg/kg-bw/day, the highest dose tested.

In a prenatal developmental toxicity study in rabbits, the LOAEL for maternal toxicity was 500 mg/kg-bw/day (the lowest dose tested) based on increased incidence of clinical signs. The NOAEL for developmental toxicity was > 2000 mg/kg-bw/day, the highest dose tested.

In a prenatal developmental toxicity study in rabbits, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg-bw/day, the highest dose tested.

Basis for Conclusions

In a range-finding study (Study # 438-033, October 27, 1981; Chlorinated Paraffin Consortium), in pregnant Charles River COBS rats administered LCCP (22-26 carbons, 43 wt% Cl) via gavage at dose levels of 0, 3000, and 5000 mg/kg-bw/day on GD 6-19, the only effects reported occurred in dams and consisted of a slight increased incidence in anogenital matting during the latter portion of the treatment period at 5000 mg/kg-bw/day. No adverse treatment-related effects were reported in offspring at doses up to 5000 mg/kg-bw/day.

Based on the findings of the range-finding study, four groups of 25 pregnant Charles River COBS CD rats were administered LCCP (22-30 carbons, 70 wt% Cl) via gavage at doses of 0, 500, 2000, and 5000 mg/kg-bw/day on GD 6-19 (Study # 438-045, April 11, 1984; Chlorinated Paraffin Consortium). Unmated males and females were individually housed and acclimated for 15-days in an environmentally controlled room. At the end of the acclimation period, all animals were weighed and subjected to a detailed physical examination. One female and one male rat were placed together for mating. Confirmation of mating was based on evidence of a copulatory plug. The day mating was confirmed was designated as day 0 of gestation. Test article was administered to pregnant females orally by gavage as a single daily dose on GD 6-19. During treatment, pregnant females were observed daily for mortality and clinical signs of toxicity. Any females not surviving to scheduled sacrifice were necropsied. Body weights were recorded on GD 0, 6, 9, 12, 16, and 20. All females were sacrificed on GD 20 and the uterus and ovaries excised for examination. The number and location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed. The abdominal and thoracic cavities underwent gross examination. Maternal tissues were preserved for future histopathological analysis. Fetuses were weighed, sexed, tagged and examined for external malformations and variations, including the palate and the eyes. The fetuses underwent visceral and skeletal examinations for malformations and developmental variations.

No adverse treatment-related effects were reported in the dams or offspring at doses up to 5000 mg/kg-bw/day. The NOAEL for both maternal and developmental toxicity was 5000 mg/kg-bw/day, the highest dose tested.

In a range-finding study (Study # 438-018, October 27, 1981; Chlorinated Paraffin Consortium) in pregnant Dutch Belted rabbits administered LCCP (22-26 carbons, 43 wt% Cl) via gavage at doses of 0, 500, 1000, 2000, 3000, and 5000 mg/kg-bw/day on GD 6-27, a slight decrease in the amount of feces and a slight increase in matting and/or staining of the haircoat was reported in the dams at > 3000 mg/kg-bw/day. No other effects were reported. Observations of offspring did not appear to be included.

Based on the results of the range-finding study, four groups of 16 Dutch Belted pregnant rabbits were administered LCCP (22-26 carbons, 43 wt% Cl) via gavage at doses of 0, 500, 2000, and 5000 mg/kg-bw/day on GD 6-27 (Study # 438-030, August 26, 1982; Chlorinated Paraffin Consortium). Unmated males and females were individually housed and acclimated for 9-weeks in an environmentally controlled room. As a result of a positive finding for parasites in stool samples collected during acclimation, all rabbits received sodium sulfamethazine in their drinking water for 19 days during the acclimation period. This treatment was terminated 5 weeks prior to study initiation and only rabbits testing negative for parasites were placed on study. At the end of the acclimation period, all animals were weighed and subjected to a detailed examination. Females were impregnated via artificial insemination. Immediately after insemination, ovulation was induced via an injection of chorionic gonadotropin in a marginal ear vein. Semen was collected from males of proven fertility and evaluated for motility. The day of artificial insemination was designated as day 0 of gestation. During treatment, pregnant females were observed for mortality and clinical signs of toxicity. Body weights were recorded on GD 0, 6, 12, 18, 24, and 28. Any females not surviving to scheduled sacrifice were necropsied. On GD 28, all surviving females were sacrificed and the uterus and ovaries excised for examination. The location and number of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed. The thoracic and abdominal cavities underwent gross examination. Pooled samples of abdominal adipose tissue from 3 dams were frozen for future analysis. Each fetus was sexed, weighed, and examined for external malformations and variations, including the palate and the eyes, as well as visceral and skeletal examinations for malformations and developmental variations, including examination of the brain and the heart.

A dose-related trend in an increased incidence in soft stool and/or anogenital matting or staining was observed in the dams beginning at the low-dose group. Three dams aborted and were sacrificed during treatment; one in the mid-dose group, and two in the high-dose group. Increases in postimplantation loss and corresponding decreases in viable fetuses, and increases in late resorptions were reported at the high-dose group, however, these effects were not reported as being statistically significant.  No signs of treatment-related developmental toxicity were reported in the offspring at < 5000 mg/kg-bw/day, although the sample size in the high-dose group was limited, precluding any definitive conclusions. Therefore, the LOAEL for maternal toxicity was 500 mg/kg-bw/day (the lowest dose tested), based on increased incidence of clinical signs. The NOAEL for developmental toxicity was > 2000 mg/kg-bw/day.

In a range-finding study (Study # 438-038, November 1, 1982; Chlorinated Paraffin Consortium) in pregnant Dutch Belted rabbits administered LCCP (22-30 carbons, 70 wt% Cl) via gavage at doses of 0, 2000, 3750, and 5000 mg/kg-bw/day on GD 6-27, increases in abortions, reductions in maternal body weight, and increases in post-implantation losses were reported at > 2000 mg/kg-bw/day. Therefore, a second range-finding study was conducted at dose levels of 0, 50, 200, and 1000 mg/kg-bw/day (Study # 438-040, November 4, 1982; Chlorinated Paraffin Consortium). A slight decrease in viable fetuses and a slight increase in postimplantation loss were reported at 1000 mg/kg-bw/day. No other effects were reported.

Based on the results of the range-finding studies, four groups of 16 Dutch Belted pregnant rabbits were administered LCCP (22-30 carbons, 70 wt% Cl) via gavage at doses of 0, 100, 300, and 1000 mg/kg-bw/day on GD 6-27 (Study # 438-039, July 18, 1983; Chlorinated Paraffin Consortium). Unmated males and females were individually housed and acclimated for 40-days in an environmentally controlled room. At the end of the acclimation period, all animals were weighed and subjected to a detailed examination. Females were impregnated via artificial insemination. Immediately after insemination, ovulation was induced via an injection of chorionic gonadotropin in a marginal ear vein. Semen was collected from males of proven fertility and evaluated for motility. The day of artificial insemination was designated as day 0 of gestation. During treatment, pregnant females were observed for mortality and clinical signs of toxicity. Body weights were recorded on GD 0, 6, 12, 18, 24, and 28. Any females not surviving to scheduled sacrifice were necropsied. On GD 28, all surviving females were sacrificed and the uterus and ovaries excised for examination. The location and number of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uterus was weighed. The thoracic and abdominal cavities underwent gross examination. Pooled samples of abdominal adipose tissue from 3 dams were frozen for future analysis. Each fetus was sexed, weighed, and examined for external malformations and variations, including the palate and the eyes, as well as visceral and skeletal examinations for malformations and developmental variations, including examination of the brain and the heart.

No treatment-related adverse effects were observed in the dams or the offspring at any dose level. The increases in postimplantation losses noted in the two previous range-finding studies reported above were not reproduced in the definitive study. Therefore, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg-bw/day, the highest dose tested.

 ENVIRONMENTAL MONITORING
    MCCP MONITORING DATA
 Surface Water
It is known that over time, based on their molecular weight and physicochemical properties, MCCPs in surface water will partition to suspended particulates, sediment, sludge, or soil. Reported MCCP concentrations in surface water range from < 2.50 x 10-10 to 1.49 x 10[-3] mg/L (Table_Apx D-1-1). Very little information is available on the specific sampling locations for many of the surface water measurements reported in Table_Apx D-1-1. Limited documentation is available on two of the studies (Petersen et al., 2006 and Muir, 2003). Two sources provide a review of the literature with very little details (IPCS, 1996 and EC, 2008b). Two studies do provide detailed information on the sampling approach, including location (Houde et al., 2008 and USEPA, 1988). The Petersen et al. (2006) study, which had the highest published concentration, reported results for water samples collected from different Norwegian locations. EPA/OPPT assumes that these samples were collected in non-marine waters. Three studies found were not used in this assessment (BUA, 1992; Hoechst, 1987; and Willis, 1994). However, all of the studies used in the assessment use modern analytical techniques, reference the specific CPs of interest, and provide, at a minimum, general information on the sampling location. Given the paucity of surface water data available, EPA/OPPT used measurements from the selected studies and used the minimum and maximum values in this assessment.

Measurements of dissolved (filtered) concentrations were generally non-detectable (ND) with few exceptions. Concentrations measured in surface water were largely from studies that measured total water concentrations which included MCCPs sorbed to particulates. More recent monitoring studies (Table_Apx D-1-1) have focused on measuring MCCPs in suspended solids, sediment pore water, and sediment.

Early analytical methods using thin layer chromatography (TLC) were used to measure CPs in surface water. However, this method has poor sensitivity and reproducibility, and provide false negative results. Current methods of quantification using gas or liquid chromatography coupled with a range of detectors (i.e., mass spectrometry; MS) are more reliable. Nearly all of the water concentrations were measurements taken at a single point in time (i.e., the samples were not time series samples). Absent more extensive monitoring data, EPA/OPPT assumed that the available data could be extrapolated to longer time periods for determination of a chronic exposure concentration.

MCCP concentrations in surface water, reported in Table_Apx D-1-1, rely on test methods that filtered or pre-filtered samples before they were analyzed, which can underestimate environmental concentrations. Where appropriate, reported values were converted to a common unit, as presented in the table. For the purposes of this assessment, in the studies considered acceptable, EPA/OPPT used the lowest and highest reported concentrations (< 2.50 x 10-10 to 1.49 x 10-3 mg/L; Table_Apx D-1-1) to evaluate risks of potential concern to aquatic organisms.
Table_Apx D-1-1: Surface Water Concentrations of MCCPs, Sorted by Country
                                     Media
                                    Country
                                   Location
                            City, State or Province
                                   Comments
                            Converted Concentration
                                 Common Units
                               Analytical Method
                                  References
                                 Surface Water
                                    Canada
                                 Lake Ontario
                                    Maximum
                                 2.60x10[-9]
                                     mg/L
                                      NR
                                  EC (2008b)

                                     <
                                 2.50x10[-10]
                                     mg/L
                                  GC-HRMS-MAB
                              Houde et al. (2008)

                                     <
                                 1.00x10[-8]
                                     mg/L
                                  GC-ECNI-MS
                              Muir et al. (2003)

                                    Maximum
                                 4.70x10[-8]
                                     mg/L
                                  GC-HRMS-MAB
                              Houde et al. (2008)

                                     Mean
                                 9.00x10[-10]
                                     mg/L
                                  GC-HRMS-MAB
                              Houde et al. (2008)

                                    Germany
                            River Lech at Langsweid
                                      ---
                                 1.90x10[-4]
                                     mg/L
                                      NR
                                  IPCS (1996)

                              River Lech at Rain
                                      ---
                                 1.70x10[-4]
                                     mg/L
                                      NR
                                  IPCS (1996)

                           River Lech at Gersthofen
                                      ---
                                 9.00x10[-5]
                                     mg/L
                                      NR
                                  IPCS (1996)

                            River Lech at Augsburg
                                     <
                                 2.50x10[-5]
                                     mg/L
                                      NR
                                  IPCS (1996)

                           River Danube at Marxheim
                                      ---
                                 7.00x10[-5]
                                     mg/L
                                      NR
                                  IPCS (1996)

                                     <
                                 3.00x10[-5]
                                     mg/L
                                      NR
                                  IPCS (1996)

                                    Norway
                                      NR
                                      ---
                                 1.49x10[-3]
                                     mg/L
                                  GC-ECNI-MS
                            Petersen et al. (2006)

                                United Kingdom
                              Multiple locations
                                     <
                                   1x10[-4]
                                     mg/L
                                  GC-ECNI-MS
                            Nicholls et al. (2001)

                                 United States
                               Sugar Creek, Ohio
                                     <
                                 7.50x10[-5]
                                     mg/L
                                  GC-ECNI-MS
                                 USEPA (1988)

                           Central European Country
                                      NR
                                     <
                                 5.00x10[-5]
                                     mg/L
                                  GC-ECNI-MS
                                Coelhan (2010)
NR: Not recorded. Location description was not provided in the study.
--: Single sample value reported above the detection limit; therefore, no data qualifier required.
GC-HRMS-MAB: Gas chromatography-high resolution mass spectrometry with metastable atom bombardment ionization
GC-ECNI-MS: Gas chromatography in combination with electron capture negative ion mass spectrometry
Notes:
1. All values provided in the table above represent total MCCP and not individual MCCP isomers