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Effects on fertility

Additional information

In a one-generation study (Stamp, 2006) [CXR, 2006] conducted according to OECD Guideline 421, and to GLP, groups of 12 male and 12 female CD-strain (Sprague-Dawley) rats (F0) were fed a diet containing 0, 300, 600 or 1200 ppm Cereclor S52 (a C14-17 chlorinated paraffin; 52% chlorinated) for 4 weeks before pairing, and throughout pairing, gestation and lactation. F0 males were killed after 9 weeks of treatment (day 4 of lactation), whilst F0 females were allowed to litter and rear their offspring and were killed on day 21 of lactation (about 11-12 weeks of treatment). Additional groups of control and top dose F0 animals (5/group) were included to furnish blood, liver and milk samples for further analysis. The calculated intakes of Cereclor S52 during the various stages of the study were: F0 males pre-pairing 0, 21, 44 and 84 mg/kg bw/day; F0 females pre-pairing 0, 23, 47 and 99 mg/kg bw/day; F0 females during gestation 0, 25, 49 and 104 mg/kg bw/day and F0 females during lactation 0, 64, 121 and 212 mg/kg bw/day, for the control, 300, 600 and 1200 ppm dose groups, respectively. MCCP did not affect clinical condition, body weight gain, food intake, oestrous cycle length, mating performance, pre-coital interval, fertility, gestation length, the number of implantations, litter size, sex ratio, or offspring survival, clinical condition, body weights, body weight gains to weaning, tissue macropathology or liver weights. The 1200 ppm F0 females had marginally higher absolute and relative mean liver weights. The no-observed-adverse-effect level (NOAEL) for fertility in this study was 1200 ppm, equivalent to a parental intake of Cereclor S52 of approximately 100 mg/kg bw/day prior to giving birth.

 

In a range-finding study (IRDC, 1985), groups of 5 male and 10 female Charles River COBS CD rats were fed Cereclor S52 in the diet at concentrations of 0, 100, 1000 and 6250 ppm for 28 days prior to mating, during mating and (in females only) up to post-natal day 21. The average doses were 0, 6, 62 or 384 mg/kg bw/day for males and 0, 8, 74 or 463 mg/kg bw/day for females. Five male and 10 female pups selected randomly from each group were fed the same diet as their parents from weaning up to 70 days of age. One litter from each group was killed on lactation days 6 (high dose) and 7 (controls); the remaining pups were sacrificed on lactation day 21. Cereclor S52 did not affect F0 survival, the microscopic appearance of selected tissues of the F0 females (kidneys, lungs, ureter and urinary bladder) or any of a number of reproductive and litter parameters. Blood samples were collected for haematological analysis from F1 pups at various timepoints. At birth, pup survival was similar in all dose groups. However, pup survival was significantly decreased during lactation in the top-dose group (probably as a result of internal haemorrhaging) and no pups survived until weaning. Pup survival was 11% lower in the mid-dose group and, although not statistically significant, is considered to be of toxicological importance. Growth was reduced (dose-related, but not statistically significant) in F1 pups in the mid- and high-dose groups on lactation days 7, 14 and 21, and in the low and mid-dose groups post-weaning. Apart from reduced growth, no adverse effects were seen in the low-dose group. Decreased activity and swollen and dark or black eye(s) were observed in a few pups in one or two mid- and high-dose litters. Haematological analyses revealed reductions in erythrocyte counts, haemoglobin concentration and haematocrit in a single litter at the top dose on lactation day 6 (compared with day 7 controls). At necropsy, there were dose-related, but not statistically significant, increases in the occurrence and severity of subcutaneous haematoma, pallor, blood around the orifices, pale liver, kidneys, lungs and spleen and blood in the cranial cavity and brain in mid- and high-dose pups. Haematoma was noted in all of the high-dose litters. In this range-finding study, no adverse effects were seen on fertility following administration of Cereclor S52 to rats in the diet at up to approximately 400 mg/kg bw/day. Significant effects were seen in the developing offspring during the lactational period (prior to full weaning) in the mid- and high-dose groups, suggesting a study NOAEL for the offspring (as a maternal dose) of approximately 8 mg/kg bw/day.

 

In a study (Barton and Daley, 2004) [CXR, 2004] designed to assess whether MCCPs transferred through breast milk disrupt the pups’ blood clotting system, five out of 32 dams given Cereclor S52 at about 538 mg/kg bw/day in the diet for 4 weeks prior to mating, and throughout mating and gestation, died while giving birth. Excessive bleeding in the clinical/necropsy findings of these dams and decreased maternal blood levels of vitamin K indicate that these deaths were probably due to haemorrhaging and not a direct consequence of parturition.

 

With regard to effects upon fertility, no information is available in humans.

 

No multi-generation studies have been conducted on MCCPs. However, C14-17 chlorinated paraffins have not shown any effects on fertility or development in reliable one-generation reproduction studies and conventional teratology studies, respectively. MCCPs, and indeed SCCPs and LCCPs, are not anticipated to be germ cell mutagens. No adverse effects have been observed on the reproductive organs of laboratory animals in repeated dose toxicity studies with MCCPs or lifetime studies with SCCPs. The observed internal haemorrhaging following exposure to MCCPs would appear to be a repeated dose effect to which newborns during lactation, and possibly pregnant females at the time of parturition, are particularly susceptible. As the overall NOAEL identified from the repeated dose toxicity studies (23 mg/kg bw/day) is significantly lower than that identified in the one-generation studies (47 mg/kg bw/day, as a maternal dose), it is anticipated that the NOAEL from the repeated dose studies is sufficient to protect the breast feeding offspring from the potential internal haemorrhaging effects of MCCPs at 74 mg/kg bw/day (a maternal dose). Overall, on the basis both of scientific and animal welfare grounds, a multi-generation study with MCCPs is not considered a high priority for future work.


Short description of key information:
In two reliable one-generation studies, no effects on fertility were seen when male and female rats were administered Cereclor S52 (a C14-17 chlorinated paraffin; 52% chlorinated) in the diet at up to approximately 100 and 400 mg/kg bw/day (the highest tested doses) for 4 weeks prior to mating, and throughout mating and (in females only) gestation (and lactation). In an additional study, five out of 32 dams given about 540 mg Cereclor S52/kg bw/day in the diet for 4 weeks prior to mating, and throughout mating and gestation, died while giving birth. No information is available on MCCP fertility effects in humans.

Effects on developmental toxicity

Description of key information
In two reliable teratology studies, no adverse effects on foetal development were seen when female rats or rabbits were administered Cerelor S52 (a C14-17 chlorinated paraffin; 52% chlorinated) by gavage during days 6-19 and 6-27 of gestation at up to approximately 5000 and 100 mg/kg bw/day (the highest tested doses), respectively. However, dietary exposure of rats to Cereclor S52 at 74 mg/kg bw/day and above has produced internal haemorrhaging and deaths in neonatal pups, an effect not seen (in another study) at 47 mg Cereclor S52/kg bw/day. No information is available on developmental effects in humans.
Additional information

In a good quality-study, to GLP, groups of 25 mated female Charles River CD rats received 0, 500, 2000 or 5000 mg/kg bw/day of Cereclor S52 in corn oil by oral gavage on gestational days 6-19 with sacrifice on day 20 (IRDC, 1984). The numbers and location of viable and non-viable foetuses, resorption sites and total number of implantations and ovarian corpora lutea were determined. All foetuses were examined for external malformations, and one half of the foetuses from each litter were then examined for visceral malformations and the other half for skeletal malformations. Clinical signs of maternal toxicity were seen at 2000 and 5000 mg/kg bw/day, comprising wet and/or matted fur in the anogenital region (with red or yellow staining) and an increased incidence of soft stool. Treatment did not affect the uterine parameters examined, pup weights or the incidence of foetal malformations. In this study, no developmental effects were observed at dose levels up to 5000 mg/kg bw/day (the highest tested dose), and this can therefore be considered as the study NOAEL for these effects (IRDC, 1984).

In a good-quality study, to GLP, groups of 16 artificially-inseminated female rabbits were given Cereclor S52 by oral gavage in corn oil at dose levels of 0, 10, 30 and 100 mg/kg bw/day on gestational days 6 to 27 and sacrificed on gestational day 28 (IRDC, 1983). The dams were examined for the numbers and positions of viable and non-viable foetuses, resorption sites, and total numbers of implantations and ovarian corpora lutea. All foetuses were examined for external, visceral and skeletal malformations. No significant treatment-related mortalities or clinical signs of toxicity were seen in the dams and no treatment-related malformations were seen in the foetuses. The few abortions (1, 2 and 2 in the 0, 30 and 100 mg/kg bw/day dose groups respectively) and the increased number of viable foetuses in the 30 mg/kg bw/day dose group were not considered to be of toxicological significance. In this study, no developmental effects were observed at dose levels up to 100 mg/kg bw/day (the highest tested dose), and this can therefore be considered as the study NOAEL. A limitation of this study was that it was not conducted up to a maternally toxic dose (IRDC, 1983).

                                                              

In relation to developmental effects, there are no data available in humans.

 

Justification for classification or non-classification

No developmental effects were seen when rats and rabbits were repeatedly dosed with MCCP during pregnancy at up to 5000 and 100 mg/kg bw/day, respectively. In one-generation reproductive toxicity studies, fertility was unaffected but pup mortality was increased (due to internal haemorrhaging) during the lactation period. Further investigations suggest that this is probably due to a combination of (a) reduced vitamin K in the breast milk due to MCCP toxicity in the dams, (b) the pups innate poor ability to generate vitamin K and (c) direct toxicity due to MCCP transfer into breast milk.

 

A NOAEL of 47 mg/kg bw/day (as a maternal dose) was established for pup mortality. Therefore, MCCPs do not meet the classification requirement for developmental or reproductive toxicants under EU DSD or CLP regulations.

 

MCCPs have been shown to cause an adverse effect (internal haemorrhaging leading to death) in the offspring of dams receiving MCCPs in their diet. It has been shown, from cross-fostering studies, that this effect is mediated via the mother’s milk.

 

Mechanistic studies have shown that the effect is due to the fact that Vitamin K levels in the pups are compromised. The available evidence suggests that this situation arises because MCCPs inhibit the absorption of Vitamin K from the GI tract, resulting in very low Vitamin K levels in the blood and liver of the dams, which in turn results in low Vitamin K levels in their milk. These effects occur after the administration of high dietary doses of MCCPs to the female rats, a situation that would not arise under any reasonably foreseeable circumstances of human exposure.

 

The criteria for the assignment of R64 (May cause harm to breast-fed babies) under Annex VI of Directive 67/548/EEC (DSD) are as follows:

 

Substances which are classified as toxic to reproduction and which also cause concern due to their effects on lactation should in addition be labelled with R64. For the purpose of classification, toxic effects on offspring resulting only from exposure via the breast milk, or toxic effects resulting from direct exposure of children will not be regarded as 'Toxic to reproduction`, unless such effects result in impaired development of the offspring. Substances which are not classified as toxic to reproduction but which cause concern due to toxicity when transferred to the baby during the period of lactation should be labelled with R64. This R-phrase may also be appropriate for substances which affect the quantity or quality of the milk. R64 would normally be assigned on the basis of:

(a) toxicokinetic studies that would indicate the likelihood that the substance would be present in potentially toxic levels in breast milk; and/or

(b) on the basis of results of one or two generation studies in animals which indicate the presence of adverse effects on the offspring due to transfer in the milk; and/or

(c) on the basis of evidence in humans indicating a risk to babies during the lactational period.

 

Substances which are known to accumulate in the body and which subsequently may be released into milk during lactation may be labelled with R33 and R64.

None of these criteria apply to MCCPs. As a consequence, it is inappropriate to assign R64 to MCCPs.

 

The criteria for the assignment ofH362 (May cause harm to breast-fed children)under CLP are as follows:

 

Effects on or via lactation are allocated to a separate single category. It is recognised that for many substances there is no information on the potential to cause adverse effects on the offspring via lactation. However, substances which are absorbed by women and have been shown to interfere with lactation, or which may be present (including metabolites) in breast milk in amounts sufficient to cause concern for the health of a breastfed child, shall be classified and labelled to indicate this property hazardous to breastfed babies.This classification can be assigned on the:

(a) human evidence indicating a hazard to babies during the lactation period; and/or

(b) results of one or two generation studies in animals which provide clear evidence of adverse effect in the offspring due to transfer in the milk or adverse effect on the quality of the milk; and/or

(c) absorption, metabolism, distribution and excretion studies that indicate the likelihood that the substance is present in potentially toxic levels in breast milk.

 

None of these criteria apply to MCCPs. As a consequence, it is inappropriate to assign H362 to MCCPs.