Registration Dossier

Administrative data

Endpoint:
biochemical or cellular interactions
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Good quality study, well reported

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Reference Type:
secondary source
Title:
Unnamed
Year:
2008

Materials and methods

Principles of method if other than guideline:
An investigation of the effects of feeding a C14-17 chlorinated paraffin (52% chlorination) to female rats (maintained on a normal or menadione-deficient diet) on plasma vitamin K levels, various clotting factors and hepatic cytochrome P450 induction.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cereclor S52
- Substance type: technical product
- Physical state: pale yellow viscous liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data on presence/absence of a stabiliser
- Composition of test material, percentage of components: C14-17 chlorinated paraffin (52% chlorination)
- Lot/batch No.: 10313/1
- Expiration date of the lot/batch: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Harlan UK- Age at study initiation: 6-8 weeks- Housing: On sawdust in solid-bottom polypropylene cages.- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 5 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19-23- Humidity (%): 40-70- Air changes (per hr): 14-15- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Cereclor S52 (0.395 or 0.79 ml) mixed with corn oil (9.605 or 9.21 ml respectively) VEHICLE- Justification for use and choice of vehicle (if other than water): test material solubility- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
21 days
Frequency of treatment:
Daily
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:0, 500 and 1000 mg/kg bw/dayBasis:actual ingested
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of treated animals maintained on either a normal or menadione-deficient diet.Two separate groups (normal and deficient diet) given 0.1% sodium phenobarbitone in the drinking water for 21 days.

Examinations

Examinations:
At termination, body and liver weights were recorded and blood samples were taken. These were analysed for prothrombin clotting times, clotting factors VII and X and vitamin K levels. In addition, liver microsomal fractions were prepared and analysed by SDS-PAGE and Western immunoblotting for assessment of induction of CYP2B1 and/or CYP2B2 isozymes.
Positive control:
Phenobarbitone (PB)-treated animals were included to test the hypothesis that induction of PB-type inducible enzymes may increase vitamin K metabolism

Results and discussion

Details on results:
No treatment-related deaths, clinical signs of toxicity or effects on body weight were observed, but there was a statistically significant, dose-related increase in liver weight in both the normal and the menadione-deficient diet groups (by 42% and 56% at 500 and 1000 mg/kg bw/day respectively for the normal diet groups, and by 42% and 49% at 500 and 1000 mg/kg bw/day respectively for the menadione-deficient diet groups). Liver weights for the phenobarbitone (PB)- treated animals were not markedly different from their respective control groups.Factor X levels were unaffected by treatment for both dietary regimes, and plasma vitamin K levels were lower (by 34%) only in the high dose animals fed menadione-deficient diet. A statistically significant, dose-related decrease (by 18% and 42% at the low and high dose respectively) in Factor VII levels was observed in the treated animals fed normal diet. A marked decrease in Factor VII levels was also seen in both control and treated animals fed menadione-deficient diet (by 25%, 24% and 44% of the normal diet control group levels at 0, 500 and 1000 mg/kg bw/day respectively). Conversely, PB-treated animals fed normal and deficient diets showed an increase in Factor VII levels. Prothrombin clotting times were slightly statistically significantly decreased (by 12%) but only at the low dose in the normal diet group.The results of the Western immunoblot analysis showed that the expression of CYP2B1 and CYP2B2 isozymes was induced at both MCCP dosages, at similar levels in the normal and in the menadione-deficient diet groups. The extent of induction by MCCPs was also similar to that observed with PB administration.

Applicant's summary and conclusion

Conclusions:
Administration of Cereclor S52 (a C14-17 chlorinated paraffin; 52% chlorination) by stomach tube to female Sprague-Dawley rats at dose levels up to 1000 mg/kg bw/day for 21 days had no significant effect on the blood clotting system. However, Cereclor S52 appears to cause induction of CYP2B1 and CYP2B2 isozymes in both the normal and menadione-deficient diet groups.
Executive summary:

In a good quality study, groups of 6 female adult Sprague-Dawley rats, fed a normal or a menadione-deficient diet, were given Cereclor S52 (a C14-17 chlorinated paraffin; 52% chlorination) at 0, 500 or 1000 mg/kg bw/day by oral gavage for 21 days. In addition, two groups of 6 female rats, one maintained on the normal diet and one on menadione-deficient diet, were treated for 21 days with 0.1% phenobarbitone (PB, an inducer of liver cytochrome P450 enzymes) in drinking water (equivalent to a dose of 20 mg/kg bw/day). At termination, body and liver weights were recorded, and blood samples were taken. The blood samples were analysed for prothrombin clotting times, clotting factors VII and X and vitamin K levels. In addition, liver microsomal fractions were prepared and analysed by SDS-PAGE and Western immunoblotting for assessment of induction of CYP2B1 and/or CYP2B2 isozymes.

In Cereclor S52 treated animals, no deaths, clinical signs of toxicity or effects on body weight were observed, but there was a statistically significant dose-related increase in liver weight in both the normal and the menadione-deficient diet groups. Factor X levels were unaffected by treatment with MCCPs for both dietary regimes, and plasma vitamin K levels were lower (by 34%) only in the high dose animals fed the deficient diet. A statistically significant, dose-related decrease in Factor VII levels was observed in the Cereclor S52-treated animals fed normal diet and in both control and Cereclor S52-treated animals fed menadione-deficient diet. Prothrombin clotting times were slightly decreased (by 12%) but only at the low dose in the normal diet group. The results of the Western immunoblot analysis showed that the expression of CYP2B1 and CYP2B2 isozymes was induced at both MCCPs dosages, at similar levels in the normal and in the menadione-deficient diet groups.

In PB-treated animals, liver weights were not markedly different from their respective control groups, but, in contrast to MCCP treatment, Factor VII levels were increased in animals fed both the normal and deficient diets. The extent of induction of CYP isozymes was similar to that observed with MCCP.

According to the draft RAR (EU, 2008), although the administration of Cereclor S52 to adult female rats at dose levels up to 1000 mg/kg bw/day caused a significant reduction in Factor VII in normal diet animals this was not of a sufficient magnitude to cause a biologically significant increase in prothrombin clotting times, and therefore MCCPs are without effect on the blood clotting system of rats.