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Administrative data

Description of key information

There are no data available in humans or laboratory animals relating to repeated inhalation or dermal exposure, or human data following oral exposure. However, a number of reliable studies have investigated the repeated dose oral toxicity of C10-13 chlorinated paraffins (58 and 60% chlorination) in rodents and this has allowed the identification of a human relevant NOAEL of 100 mg/kg bw/day from a well-conducted 90-day dietary GLP study in rats administered a C10-12 chlorinated paraffin (58% chlorination), with kidney effects seen at a higher dose in this study and others.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

No relevant data in humans currently available on the repeated dose effects of SCCPs.

Repeated dose oral toxicity in rats

In a 13-week GLP study, similar to OECD Guideline 408, groups of male and female Fischer 344 rats (15/sex/dose) were given a C10-12 chlorinated paraffin (58% chlorination) in the diet at nominal dose levels of 10, 100 and 625 mg/kg bw/day (actual intakes were 10, 101 and 654 mg/kg bw/day for males and 10.3, 102 and 613 mg/kg bw/day for females). During the study, animals were observed for clinical signs of toxicity. Food and water consumption was determined and haematological and urinary analyses performed. At the end of the feeding period, animals were weighed and a range of organs examined for gross and histopathological changes. Livers were also recovered for biochemical investigations. No deaths occurred and no clinical signs of toxicity (other than slight skin atonia in top-dose males) were observed during the study. A slight reduction in body weight gain (9% compared to controls) was noted in top-dose males after 13 weeks and a reduction in water consumption was seen in top-dose males and females (11 and 20%, respectively). This was associated with a decrease in urine volume and an increase in specific gravity. Increases in blood total protein (13%), cholesterol (54%), glucose (20%) and urea nitrogen (44%) were also seen in top-dose animals and in glucose (20%), cholesterol and urea nitrogen in mid-dose animals. Statistically significant increases in liver (about 20 and 140%) and kidney (10 and 30%) weights were seen in mid- and top-dose animals, respectively, and in thyroid weight (about 32%) in top-dose animals only (these percentages represent average increases when combining absolute and relative weight changes). Histopathological changes seen included hepatocellular hypertrophy in both mid- and top-dose males and females, mild chronic nephritis in mid- and top-dose males and renal tubule pigmentation in top-dose females, and thyroid hypertrophy and hyperplasia in mid-dose males and top-dose males and females. The no-observed-effect level (NOEL) in the rat for the C10-12 chlorinated paraffin (58% chlorinated) investigated in this study is 10 mg/kg bw/day (IRDC, 1984a). However, the changes in the kidney are of doubtful toxicological significance. Liver weight, histopathology and enzyme changes reflect xenobiotic metabolism and peroxisome prolifferation and are not considered to be of toxicological significance to humans. Similarly, effects on the thyroid are not considered to be relevant to humans [see mechanistic data below]. Other signs of toxicity were noted at doses greater than 100 mg/kg bw/day. Therefore, a value of 100 mg/kg bw/day will be taken forward to the risk characterisation as the human relevant no-observed-adverse-effect level (NOAEL; see overall summary below).

In a 13-week GLP study, similar to OECD Guideline 408, groups of male and female Fischer 344 rats (15/sex/dose) were given a C10-12 chlorinated paraffin (58% chlorination) by oral gavage (in corn oil) at nominal dose levels of 10, 100 and 625 mg/kg bw/day. During the study, animals were observed for clinical signs of toxicity. Food and water consumption was determined and haematological and urinary analyses performed. At the end of the dosing period, animals were weighed and a range of organs examined for gross and histopathological changes. Livers were also recovered for biochemical investigations. No deaths occurred and no clinical signs of toxicity (other than slight skin atonia in top-dose males and females) were observed during the study. A slight, non-significant reduction (about 4.5% less than controls) in body weight gain was noted in top-dose males after 13 weeks and a slight, significant increase in top-dose females (5% greater than control). An increase in water consumption was seen in top-dose males and females (22 and 31%, respectively). This was associated with an increase in urine volume and a decrease in osmolality and specific gravity. Decreases in serum cholesterol were seen in mid- and top-dose males only (to 76 and 61% of control) and increases in serum protein and phosphorous and decrease in alkaline phosphatase activity detected in top-dose males and females. Statistically significant increases in liver (about 26 and 123%) and kidney (about 12 and 28%) weights were seen in mid- and top-dose animals, respectively, and in thyroid weights (about 25%) of the top-dose males only. These organ weight changes were associated with histopathological changes. Hepatocellular hypertrophy was noted in both mid- and top-dose males and top-dose females, whereas kidney lesions (nephrosis in males and pigmentation in females) and thyroid lesions (follicular cell hypertrohy and hyperplasia in both sexes) were only found in top-dose animals only. The NOEL in the rat for the C10-12 chlorinated paraffin (58% chlorination) investigated in this study is 10 mg/kg bw/day (IRDC, 1984b). [See discussion below for the relevance to humans of these effects].

In a NTP 13-week study, similar to OECD Guideline 408, groups of 10 male and 10 female Fischer 344 rats were given a C12 chlorinated paraffin (60% chlorinated) daily by gavage (in corn oil) at target dose levels of 313, 625, 1250, 2500 and 5000 mg/kg bw, 5 days/week. At the end of the dosing period animals were weighed and a full range of organs examined for gross and histopathological changes in the control and top dose groups. No compound-related deaths were seen and only a slight reduction in body weight gain was seen in male rats given the top two dose levels (12 and 11% lower at 5000 and 2500 mg/kg bw/day, respectively, when compared to controls). A statistically significant dose-related increase in relative liver weights was seen for both males and females (about 25, 38, 55, 100 and 100% at 313, 625, 1250, 2500 and 5000 mg/kg bw/day, respectively) with hepatocellular hypertrophy noted in all top-dose animals examined. Nephropathy was also noted in all top dose males and in 3/10 top dose females, but also at a lesser severity in 8/10 control males (and 0/10 control females). No changes were seen in any of the other organs examined. The liver changes seen were ascribed to the peroxisome proliferating effects of SCCPs and the kidney effects to a compound-related worsening of this age-related condition. In view of the significant effects on the liver seen at the lowest dose level examined, a NOAEL could not be established in this study (NTP, 1986).

In a 14-day toxicity study (to GLP, and similar to OECD Guideline 407), groups of 5 male and 5 female Fischer 344 rats were given a C10-12 chlorinated paraffin (58% chlorinated) by oral gavage (in corn oil) at doses of 0, 30, 100, 300, 1000 and 3000 mg/kg bw/day. During the study animals were observed for mortality and clinical signs of toxicity and body weight, food and water consumptions recorded. At the end of the dosing period, surviving animals were killed and a complete post-mortem with histological examination of tissues carried out. In addition, liver microsomal protein and cytochrome P-450 content and aminopyrene N-demethylase activity was determined in all treatment groups. No treatment related deaths occurred. This animal, and a few others showed yellow staining of the anogenital region (2 males and 2 females at the top dose and 1 male at 300 mg/kg bw/day), brown staining around the mouth (1 female at 3000 mg/kg bw/day) and laboured breathing (1 female at 1000 mg/kg bw/day). Top dose animals showed a reduction in body weight gain (males 15%; females 20%) and food consumption (males 13%; females 20%) although the reduced weight gain was only significant for females. At the end of the feeding period, dose-related increases in absolute and relative liver weights were seen in males (about 20%) at 100 mg/kg bw/day and in males and females (about 20-40, 50-80 and 60-150%) at 300, 1000 and 3000 mg/kg bw/day, respectively. Top dose animals also showed a decrease in absolute and relative thymus weight (at least 50%) and ovary weights (more than 35%). Diffuse, mild hepatocellular hypertrophy was noted in 2/5 male and 2/5 female rats at 300 mg/kg bw/day and in all but one rat given 1000 and 3000 mg/kg bw/day. Other histological changes noted were not considered to be treatment-related. The biochemical and histopathological changes seen in the liver were considered by the authors to be adaptive physiological responses. A no-observed-adverse-effect level (NOAEL) in this study was considered to be 1000 mg/kg bw/day, based on effects on thymus and ovaries weights at a the top dose level (IRDC, 1981).

In a 14-day toxicity study (to GLP, and similar to OECD Guideline 407), groups of 5 male and 5 female Fischer 344 rats were fed a C10-12 chlorinated paraffin (58% chlorinated) in the diet at dose levels of 0, 900, 2700, 9100 and 27,300 ppm (equivalent to approximately 0, 100, 300, 1000 and 3000 mg/kg bw/day). During the study animals were observed for mortality and clinical signs of toxicity, and body weights and food consumption were recorded. At the end of the feeding period, animals were killed and a complete post mortem with histological examination of tissues was carried out. In addition, liver microsomal protein and cytochrome P-450 content and aminopyrene N-demethylase activity was determined in all treatment groups. No deaths or clinical signs of toxicity were seen during the treatment period. A marked reduction in food consumption and body weight gain was seen in both males and females (approximately 50% by day 14) and statistically significant and dose-related increases in absolute and relative liver weights were noted in all treatment groups (approx. 20, 50, 110 and 150-240% at 100, 300, 1000 and 3000 mg/kg bw/day, respectively, compared to controls). In all treatment groups, an increased incidence and extent of hepatocellular hypertrophy was seen as well as a dose-related increase in the hepatic microsomal examined. In females the increase in all microsomal parameters was statistically significant at 300 mg/kg bw/day and above; in males cytochrome P-450 levels were statistically significantly increased at 1000 mg/kg bw/day and above. Atrophy of the spleen, thymus and testes was seen in the top dose groups and myocardial atrophy in all of the top dose animals and 9/10 of the 1000 mg/kg bw/day animals. The no-observed-effect level (NOEL) for the test material in this study was considered to be 300 mg/kg bw/day, based on myocardial effects. However, myocardial atrophy was considered by the authors to be associated with weight loss, at least in the top dose animals. in addition, as this effect has not been reported in any of the other studies it is considered in the final RAR (EU, 2000) to "not be of toxicological significance in relation to chlorinated paraffins". Atrophy of the spleen, thymus and testes in top dose animals was also considered to be secondary to reduced food consumption.The biochemical and histological changes seen in the liver were considered by the authors to be adaptive physiological responses and other effects in top dose animals secondary to their nutritional status.

Groups of five rats of each sex were administered 0, 469, 938, 1875, 3750 or 7500 mg/kg bw of a C12 chlorinated paraffin (60% chlorination) by gavage, on 12 days over a 16 day period (NTP, 1986). Three deaths occurred in top-dose animals. All top dose animals showed diarrhoea with males and females showing a 22% and 14% inhibition in body weight gain, respectively. Male rats treated with 3750 mg/kg bw showed a 15% inhibition in body weight gain. Enlarged livers were observed in 3-5 animals in every dose group apart from the females treated with 469 mg/kg bw; however the degree of enlargement was not discussed. Histological examinations were not conducted. The liver enlargements are likely to be due to a physiological response to the demand for xenobiotic metabolism or peroxisome proliferation, neither of which are considered to be of adverse health significance to humans (see mechanistic data below). Other signs of toxicity were noted at doses greater than 1875 mg/kg/day (NTP, 1986).

In a briefly reported, unpublished study, groups of 10 male rats were administered 0 or approximately 5000 mg/kg bw/day and 10 females, 0 or approximately 2500 mg/kg bw/day of a C10-13 chlorinated paraffin (52% chlorination), by gavage, on 14 consecutive days. All treated animals showed slight piloerection during the experiment and females were "slightly" incontinent. One treated male died after nine doses. Urinalysis showed no changes compared to controls. Evidence of slight anaemia and decreased blood clotting capability were noted in treated males and females. Animals killed 24 hours after the final treatment showed marked hepatocyte enlargement, apparently associated with proliferation of smooth endoplasmic reticulum. An unspecified number of animals, killed 7 days after the final treatment, showed similar but less marked liver changes. Three males and one female also showed slightly increased splenic haemopoiesis. No further details were given according to expert review (cited in EU, 2000).

A 2-year carcinogenicity study (summarised in IUCLID Chapter 7.7) identified the liver, kidney, thyroid and stomach to be the target organs when rats were treated with a C12 chlorinated paraffin (60% chlorination) by gavage with 312 or 625 mg/kg/day (NTP, 1986).

Repeated dose oral toxicity in mice

In a 13 week NTP study, simialr to OECD Guideline 408, groups of 10 male and 10 female B6C3F1 mice were given a C12 chlorinated paraffin (60% chlorinated) daily by gavage (in corn oil) at target dose levels of 0, 125, 250, 500, 1000 and 2000 mg/kg bw, 5 days/week. At the end of the dosing period animals were weighed and a full range of organs examined for gross and histopathological changes in the control and top dose groups and in animals that died early. In addition the livers of all animals were examined for histopathological changes. No treatment-related deaths were seen, although a total of 26 mice died early due to dosing errors. A slight reduction in body weight gain was seen in male mice only given the top two dose levels (13 and 8% at 2000 and 1000 mg/kg/day, respectively). A statistically significant dose-related increase in relative liver weights was seen for both males and females (38, 64 and 140% in males at 500, 1000 and 2000 mg/kg/day respectively and 20, 41, 96 and 172% at 250, 500, 1000 and 2000 mg/kg bw/day in females). A dose-related increase in the incidence of hepatocellular hypertrophy and focal (centrilobular) necrosis was noted in both male and female mice, the incidence tending to be greater in males. Thus, hypertophy was seen in 4, 4, 10 and 8 out of 10 males/dose at 250, 500, 1000 and 2000 mg/kg bw/day resectively, with the corresponding incidences in females at these dose levels being 1, 2, 3 and 8, respectively. Similarly, the incidence of liver necrosis in males was 1, 0, 3, 2 and 7 out of 10/dose at 125, 250, 500, 1000 and 2000 mg/kg bw/day and only 3/10 at 2000 mg/kg bw/day (all other doses were free from necrosis) in females. No changes were seen in any of the other organs examined. Based on effects seen in the liver, the no-observed-adverse-effect level (NOAEL) for the C12 chlorinated paraffin (60% chlorinated) in this study is 125 mg/kg bw/day (NTP, 1986). The liver changes seen were ascribed to the peroxisome proliferating effects of SCCPs, and are unlikely to be relevant to humans. Therefore a value of 1000 mg/kg bw/day is considered the human health relevant NOAEL in mice, as general signs of toxicity were observed at doses greter than this.

Groups of five mice of each sex were administered 0, 938, 1875, 3750, 7500 or 15000 mg/kg bw of a C12 chlorinated paraffin (60% chlorination) by gavage, on 12 days over a 16 day period (NTP, 1986). Due to the large volume of material used, the top two doses were administered in two treatments, 5 hours apart. All mice that received 3750, 7500 and 15000 mg/kg/day and 6/10 receiving 1875 mg/kg/day died before the end of the study. Diarrhoea was noted in all chlorinated paraffin-treated animals apart from the lowest-dose females. Livers appeared enlarged in treated animals which survived until the end of the study. Histological examinations were not conducted (NTP, 1986).

A 2-year carcinogenicity study (summarised in IUCLID Chapter 7.7) identified the liver, kidney and thyroid to be the target organs when mice were treated with a C12 chlorinated paraffin (60% chlorination) by gavage with 125 or 250 mg/kg/day (NTP, 1986).

Repeated dermal toxicty data

The dermal irritancy to 'shorn' rat skin of various samples of C10-13 chlorinated paraffins (with extent of chlorination varying from 41-70%) was investigated. Groups of 3 female rats were given up to six repeated applications of various samples of the chlorinated paraffin, either undiluted (0.1 mL) or as a concentrated solution in a non-irritant solvent (0.1 mL). The material was applied on alternate days under an occlusive dressing for 24 h periods, after which time the test material was removed by washing. In some individual experiments, an autopsy was performed to investigate signs of systemic toxicity, but none were reported (Birtley et al. 1980).

Repeated inhalation toxicity data

In accordance with column 2 of REACH Annex VIII and IX, repeated dose toxicity testing by the inhalation route is not considered appropriate as exposure of humans to SCCPs via inhalation is unlikely taking into account their low vapour pressure and the low likelihood of exposure to aerosols, particles or droplets of an inhalable size.

Studies on mechanisms of toxicity

A number of studies are available which have been designed to investigate the possible mechanisms of the toxic effects observed in animals, in order to establish their relevance to humans.

Studies in rats

Male rats were assessed for effects in the liver following treatment by gavage with 0, 10, 50, 100, 250, 500 or 1000 mg/kg/day C10-13, 58 or 56% chlorinated paraffin for 14 days (Wyatt et al., 1993; cited in EU, 2000). Livers were removed, weighed, homogenised and an assay was performed for peroxisomal fatty acid B-oxidation, which is a marker for peroxisomal proliferation. With the 58% chlorinated paraffin, both absolute and relative liver weights showed a dose-related increase (from 28 to 60%) with increases being statistically significant with 250 mg/kg/day and above.

Oxidase activity also showed a statistically significant increase with 250 mg/kg/day and above, reaching an almost 3-fold increase with the top-dose. With the 56% chlorinated paraffin, absolute and relative liver weights showed a dose-related increase (from 20 to 77%) with increases being statistically significant with 100 mg/kg/day and above. Oxidase activity again showed a statistically significant increase with 250 mg/kg/day and above, reaching an almost 3-fold increase with the top-dose. Top-dose animals in this study were also assessed for effects on the thyroid, by analysing blood samples for thyroid stimulating hormone (TSH), and total and free T3 and T4. Uridine diphosphate glucuronosyl (UDPG) -transferase activity, a liver enzyme involved in the excretion of T4, was measured in liver microsomes. With both chlorinated paraffins, free and

total T4 levels were decreased by 30-40% and 2-fold increases were noted in liver microsomal UDPG-transferase activity and plasma TSH levels. There were no changes in T3 levels.

In a similar study, male and female rats were treated by gavage with 0, 313, 625 or 1000 mg/kg/day 58% chlorinated paraffin for 0, 15, 29, 57 or 91 days (Elcombe et al., 1994; cited in EU, 2000). The liver, thyroid and kidney were examined histologically and as above, blood samples were analysed for total and free T4 and TSH and liver homogenates for UDPG-transferase activity. Seven days before sacrifice on days 29 and 91, animals were subcutaneously implanted with minipumps containing bromodeoxyuridine. Statistically significant increases in relative liver weight, of approximately 50 and 75% were noted with doses of 313 and 625 mg/kg/day, respectively. These increases were noted at the first kill (15 days) and did not continue to increase further at the later sacrifice times (absolute liver weights were not reported). Peroxisomal B-oxidation was also noted to show a dose-related and statistically significant increase with doses of 313 and 625 mg/kg/day from day 15, and like the relative liver weights, did not continue to increase at later sacrifice times. Liver weights and B-oxidation were apparently not recorded in the top-dose animals. It was claimed that hepatic peroxisome proliferation was also evidenced ultrastructurally, although no details were presented.

UDPG-transferase activity also showed a dose-related and statistically significant increase of at least 150%, with doses of 313 and 625 mg/kg/day from day 15. As with the liver weights and B-oxidation, the UDPG-transferase activity did not continue to increase further at the later sacrifice times. Statistically significant decreases (up to approximately 50%) in total and free plasma T4 were noted with 1000 mg/kg/day, at all time points. Decreases in total and free plasma T4 were also noted with doses of 625 and 313 mg/kg/day, although these decreases were not always statistically significant (generally only significant on days 15 and 57). With 1000 mg/kg/day, "marked" increases in plasma TSH were observed from day 8 to 15, with non-statistically significant increases being noted at the later time points. Thyroid follicular

cell hypertrophy was also apparently noted with 313 mg/kg/day and above at all time points and hyperplasia at days 56 and 91, although no further details were given. A statistically significant increase in replicative DNA synthesis in thyroid cells was also noted on day 91

with 313 mg/kg/day and above. Renal tubular eosinophilia, increasing in intensity with time, was noted from day 15 in male rats treated with 313 and 625 mg/kg. From day 29 increasing numbers of males showed initially focal and then multifocal areas of basophilia. No kidney effects were noted in the female rats. Hyaline droplet formation was not confirmed by immunocytochemical techniques (personal communication, ICI, 1995; cited in EU, 2000), however the response was indicative of this male rat specific phenomenum. Male and female rats were treated by gavage with 0 or 1000 mg/kg/day C10-13, 56 or 58% chlorinated paraffins for 14 days (Elcombe et al., 1995; cited in EU, 2000). Microscopic examination of the

liver showed hepatocyte hypertrophy and proliferation of peroxisomes and smooth endoplasmic reticulum. Morphometric analysis confirmed "marked" peroxisome proliferation with both chlorinated paraffins, with statistically significant increases being noted in peroxisome volume density. Absolute liver weights were not reported. Relative liver weights and total cytochrome P450 levels showed at least 2-fold increases compare to controls and peroxisomal B-oxidation showed 3 to 8-fold increases with the effect being greater in males. Another two studies investigated the early changes in the liver and thyroid when male and female rats were treated by gavage with 0 or 1000 mg/kg/day C10-13, 58% chlorinated paraffin for 1, 2, 4, 7, 15 or 28 days (ICI Draft paper 1 and 2; cited in EU, 2000). Histopathological examination revealed hepatocyte eosinophilia on day 1, which was followed by centrilobular and pan-lobular hypertrophy which is taken to be indicative of an increase in the number of peroxisomes. The first biochemical change to be detected was a statistically significant increase in hepatic peroxisomal B-oxidation on day 2 in the males and day 4 in the females, which reached a maximum by days 7 and 15 in the males (approximately 3-fold increase) and females (approximately 9-fold increase) respectively. This was accompanied by a progressive increase in absolute and relative liver weight which was small but statistically significant from day 2 in the males and day 4 in the females (increases of approximately 10% on day 2, 60% on day 4). Thyroid follicular cell hypertrophy was noted on day 4 in both sexes and increased with time. In the males liver UDPG-transferase activity was consistently higher than control values from

day 2 onwards, although not statistically significant until day 4. The activity in the females showed small non-statistically significant increases on days 4 to 15. Free and total T4 was reduced by up to approximately 50% in both sexes throughout the study from day 1. Reductions of approximately 30% in plasma concentrations of free and total T3 were seen in the males, during the first 4 days of the study. T3 levels were not measured in the females. The changes in the T4 levels noted to occur before changes in UDPG-transferase activity, may be a

reflection of the sensitivity of the respective assays used. TSH levels were elevated in males and females throughout the study, although the increases were not always statistically significant.

In a poorly reported intraperitoneal study, rats were administered 0 or 1000 mg/kg/day C10-13, 49, 59 or 71% chlorinated paraffin on days 1 and 4 or days 1, 4 and 6 (Nilsen et al., 1981; cited in EU, 2000). With all three chlorinated paraffins, an increase in the occurrence and size of hepatocellular cytoplasmic lipid droplets was noted. The 49% chlorinated paraffin also produced a 20 to 30% increase in the size of the hepatocytes on days 5 and 7, a proliferation of smooth endoplasmic reticulum, a "moderate" increase in the numbers of mitochondria and an increase in the size and number of peroxisomes. It is not clear whether these effects, including the peroxisome proliferation, were not noted with the higher chlorinated paraffins or if such effects were not investigated.

Studies in mice

Male mice were assessed for effects in the liver following treatment by gavage with 0, 10, 50, 100, 250, 500 or 1000 mg/kg/day C10-13, 58 or 56% chlorinated paraffin for 14 days (Wyatt et al., 1993; cited in EU, 2000). Livers were removed, weighed, homogenised and assays performed for

peroxisomal fatty acid B-oxidation. With the 58% chlorinated paraffin, absolute and relative liver weights showed a dose-related increase, with increases (from 23 to 89%) being statistically significant from 500 and 250 mg/kg/day, respectively. The oxidase activity showed a statistically significant increase with 250 mg/kg/day and above, although a non-statistically significant increase of 67% above the control value was noted with 100 mg/kg/day. Increases in oxidase activity reached a 7-fold increase with the top dose. With the 56% chlorinated paraffin, absolute and relative liver weights showed a dose-related increase, with increases (from 26 to 85%) being statistically significant with 100 mg/kg/day and above. Oxidase activity showed a statistically significant increase with 250 mg/kg/day and above, reaching a 10-fold increase with the top-dose. Male and female mice were treated by gavage with 0 or 1000 mg/kg/day C10-13, 56 or 58% chlorinated paraffins for 14 days (Elcombe et al., 1995; cited in EU, 2000). Microscopic examination of the liver showed hepatocyte hypertrophy and smooth endoplamsic reticulum and peroxisome proliferation. Morphometric analysis confirmed "marked" peroxisome proliferation with both chlorinated paraffins, with statistically significant increases being noted in peroxisome volume density. Compared to controls, relative liver weights and total cytochrome P450 levels were increased by 40 to 80% respectively. Absolute liver weights were not reported. Peroxisomal B-oxidation showed 4 to 6-fold increases.

Studies in guinea-pigs

Male guinea-pigs were treated by gavage with 0, 500 or 1000 mg/kg/day 58% chlorinated paraffin for 14 days (Elcombe et al., 1994; cited in EU, 2000). The liver and thyroid were examined histologically and as above, blood samples subjected to analysis for total and free thyroxine and TSH. No effects on thyroid homeostasis (that is, changes in thyroid hormones) were seen and no evidence of hepatic peroxisome proliferation or renal changes were noted. Liver weights were not reported.

Male guinea-pigs were treated by gavage with 0 or 1000 mg/kg/day C10-13, 56 or 58% chlorinated paraffins for 14 days (Elcombe et al., 1995; cited in EU, 2000). No treatment-related changes were observed by electron microscopy of the liver and morphometry showed no evidence of

peroxisome proliferation. Absolute liver weights were not reported. Relative liver weights showed increases of 36 to 50%; however no changes in total cytochrome P450 levels or peroxisomal B-oxidation were noted.

In a briefly reported study, guinea pigs were treated by gavage with 0, 500 or 1000 mg/kg/day C10-13, 58% chlorinated paraffin for 14 days (ICI Draft paper 3; cited in EU, 2000). This study formed part of the above study (personal communication, ICI, 1995; cited in EU, 2000). A statistically significant decrease in body weight gain of approximately 12% with both doses was noted at the end of the study. No change was noted in absolute liver weight although there was a statistically significant increase in relative liver weight (of approximately 18% with both doses). A dose-dependent loss of glycogen was detected in the livers of treated animals. There were no other histological changes in the liver, thyroid or kidney. Nor were there any changes in plasma levels of T3, T4 or TSH.

Overall assessment of mechanistic studies

The results of these mechanistic studies indicate that SCCPs produce peroxisome proliferation in rats and mice which probably underlies the liver damage observed in some prolonged exposure studies. Peroxisome proliferation has been evidenced by microscopy, morphometric analysis and marker enzyme activity. Fourteen-day studies in rats and mice have indicated a no effect level for peroxisome proliferation of 100 mg/kg bw/day. Although the threshold for the effect is the same in rats and mice, mice show a much greater peroxisome proliferation with higher doses. Peroxisome proliferation was not observed in studies in guinea pigs which are known to be insensitive to such an effect. Similarly, humans are also recognised to be insensitive to the effects of peroxisomal proliferating agents (Bentley et al., 1993, Ashby et al., 1994; cited in EU, 2000). Consequently, it can be concluded that the liver damage observed in studies in rats and mice is not relevant to human health. The only effect on the liver at doses below those producing peroxisome proliferation is small but statistically significant increases in liver weight. Such increases probably reflect increases in xenobiotic metabolism and are not considered to be of toxicological significance.

SCCPs also cause effects in the thyroid in rats and mice but not the guinea-pig. From the hepatic enzyme and hormone studies considered above, these effects appear to be due to stimulation of the thyroid via negative feed back mechanisms. The chain of events starts with a liver effect, namely an increase in UDPG-transferase. The UDPG transferase activity results in an increase in excretion of T4 and a resultant decrease in plasma T4 levels. The decrease in plasma T4 produces an increase in the release of pituitary TSH which in turn triggers a compensatory increase in the production of T4 by the thyroid. Since T4 is continually excreted and the thyroid stimulated, the increased activity in the thyroid eventually leads to hypertrophy, hyperplasia and as a consequence, a tendency to develop thyroid tumours.

It is possible that the increase in UDPG-transferase activity is a direct consequence of peroxisome proliferation or alternatively that it is triggered by the same mechanism as that producing peroxisome proliferation. However, from the evidence available, it is not clear whether or not the two are linked, although neither peroxisome proliferation nor thyroid effects (including changes in plasma T4 and TSH) were seen in studies in guinea pigs at high doses of 1000 mg/kg/day. In addition, it has been suggested that rodents are particularly susceptible to changes in the thyroid due to the absence of a T4-binding globulin which is present in humans and which has a very high affinity for T4 (Dohler et al., 1979; cited in EU, 2000). Other binding proteins are present in rodents, however their binding efficiency is considerably less than T4-binding globulin. In rodents, in the absence of T4-binding globulin, more free T4 is available for metabolism and thus excretion from the body. This would be potentiated by increased UDPG-transferase activity. Hence humans are likely to be less susceptible to changes in plasma levels of T4 and to the subsequent thyroid stimulation, seen in rats and mice in the studies above. Overall, taking into account the probable mechanisms indicated above, the apparent association with the hepatic effects observed and the difference in T4 binding between humans and rats, the effects seen in the thyroid in rats and mice are considered unlikely to be relevant to human health.

Overall summary of repeated exposure studies

There is no information available on the effects of repeated exposure to SCCPs in humans. No standard inhalation or dermal studies in animals are available, although SCCPs are likely to exert minimal systemic toxicity following dermal exposure. All available oral studies in animals were

conducted using 52 to 60% chlorinated SCCPs, and therefore it is not possible to observe directly from data whether different degrees of chlorination would alter the toxicity.

The liver and thyroid were identified as target organs in the oral studies in rats and mice. Small increases in liver weight are likely to be due to a response to xenobiotic metabolism which is not of toxicological significance. Larger increases in liver weight and hepatocelluar hypertrophy have been shown to be a reflection of peroxisome proliferation. Humans are not susceptible to peroxisome proliferation and hence the liver effects are considered not to be relevant to human health (Bentley et al., 1993, Ashby et al., 1994; cited in EU, 2000). Increases in thyroid weight and follicular cell hypertrophy have been shown to be caused by stimulation of the thyroid via a negative feedback mechanism, initiated by increased excretion and plasma depletion of T4. The depletion of T4 is a result of increased liver enzyme activity (UDPG-transferase) which may be related to peroxisome proliferation. Also humans and rodents show different T4-globulin binding characteristics which results in humans being less susceptible to plasma T4 depletion and hence to thyroid stimulation. Overall the thyroid effects seen in rats and mice are considered unlikely to be relevant to human health. Other signs of toxicity, such as reductions in body weight gain and increases in kidney weight, were observed in several 14- and 90-day studies in rats with doses greater than 100 mg/kg/day. In mice general signs of toxicity were observed in a 90-day study at doses greater than 1000 mg/kg/day. Therefore NOAELs, for effects which are considered to be relevant to human health, of 100 and 1000 mg/kg bw/day were observed in rats and mice, respectively. A NOAEL of 100 mg/kg bw/day (in rats) will be taken forward to the risk characterisation for SCCPs.

Justification for classification or non-classification

Under the EU CLP and DSD regulations, SCCPs would not be classified as a specific target organ toxicant as the observed kidney affects (considered of relevance to humans) in the 90-day oral rat studies were seen at doses significantly higher than 100 mg/kg bw/day.