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Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 December 2014 to 05 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Objective of study:
absorption
distribution
excretion
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
See Any other information for details
Qualifier:
according to
Guideline:
other: Directive 96/54/EC B.7 (OJL 248, 1996); Commission Regulation (EC) No. 440/2008, B.7.
Deviations:
yes
Remarks:
See Any other information for details
GLP compliance:
yes (incl. certificate)
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Species and strain: Crl:WI rats Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) Hygienic level: SPF at arrival, standard laboratory conditions during the study Justification of species/strain: Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies Number of animals: 8 male rats plus 2 spare animals Age of animals: Young adult rats, approx. 7 weeks old at starting Body weight at treatment: 268-284 g Acclimation period: 5 daysAnimal health: Only healthy animals were used for the test, as certified by the veterinarian Room number: 243 Cage type: The animals were kept in special metabolic cages, designed for continuous collection of urine and faeces (22 x 22 x 18 cm) Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m. Temperature: 20.2-22.6°C Relative humidity: 39-54% Ventilation: 15-20 air exchanges/hour Housing/Enrichment: Rodents were housed individually, to meet the special requirements and purpose of this study.The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phase. Diet and water supply Animals received ssniff® SM R/M "Autoclavable complete feed for rats and mice – breeding and maintenance" (Batch number: 680 2237, Expiry date: March 2015) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany). Animals received tap water from the municipal supply as for human consumption from 500 mL bottle, ad libitum. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, Address: H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives at CiToxLAB Hungary Ltd. The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.Bedding Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. “GRADE 5” produced by Johannes Brandenburg GmbH & Co. KG (Arkeburger Str. 31, DE-49424 Goldenstedt, Germany) or “Lignocel 3/4S Fasern” produced by J. Rettenmaier & Söhne GmbH & Co.KG (Address: Holzmühle 1, 73494 Rosenberg, Germany) was used in the study for this purpose. Nest building material was also provided for animals (DC Dental Central Grosshandelges. mbH, Carl-Zeiss Str. 2, D-22946 Trittau, Germany). Animal identification Each animal was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The animal number consisted of 4 digits, the first digit was the group number, the last the animal number within the group, as indicated in the Experimental design section. The cages were identified by cards holding information at least about study code, sex, dose group, cage number and individual animal number.Randomization During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight. The randomisation was checked by computer software (SPSS/PC+) using the body weight to verify the homogeneity and variability between/within the groups.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test item was administered undiluted at a constant concentration adjusting for the relative density of the test material (0.81 g/mL as provided by the Sponsor). The dose volume was 2.5 mL/kg body weight at the temperature 22 ± 3 °C. Analytical determination of the test item concentration, stability and homogeneity during treatment was not performed because of the character and the short period of study.
Duration and frequency of treatment / exposure:
The test item was administered on Day 0 by oral gavage.
Remarks:
Doses / Concentrations:The dose level was set as 2000 mg/kg bw.
No. of animals per sex per dose:
5 males in the treated (2000 mg/kg bw) group2 males in the untreated (distilled water) group
Control animals:
yes, concurrent no treatment
Positive control:
Positive control not required for this study.
Details on study design:
Justification of species/strainWistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.Rationale for dose-selection and route of administration The dose level (2000 mg/kg bw) was set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a preliminary dose range finding study of this test item conducted at the Test Facility [CiToxLAB study code 14/328-220PE]. 2000 mg/kg bw dose level is expected to be non-toxic, but high enough to allow the identification of the test item in excreta (faeces). The oral route was selected as it is a possible route of exposure to the test item in humans.
Details on dosing and sampling:
IN-LIFE PROCEDURES Clinical observations and neurological assessment Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made at least daily towards the end of the working day as practical.Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) or signs of neurotoxicity were also evaluated. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Body weight measurements Individual body weights were recorded with precision of 1 g at randomization and daily afterwards. Food consumption measurements Animal food consumption was determined by re-weighing the non-consumed diet with a precision of at least 0.1 g daily. Urine and faeces collection Urine and faeces were collected in metabolic cages for the following period from each animal: 0-6 hours post treatment 6-24 hours post treatment 24-48 hours post treatment 48-72 hours post treatment Additionally, to collect any residual test item from the metabolism bowls, wet and dry paper towels and the residual were collected on each day, and the metabolism bowl and all other relevant surfaces were washed with solvent (n-Heptane) on Day 3. Collected samples, residuals and paper wipers were stored frozen (at approximately -20 °C) until analysis. After completion of the 72 hours collection period, all animals were sacrificed.TERMINAL PROCEDURES At termination, animals were euthanised under pentobarbital anaesthesia by exsanguination. Animals were exsanguinated, blood samples for analytical purposes were taken by heart puncture into three 1.2 mL tubes containing 1.6 mg EDTA/mL blood as anticoagulant. After exsanguination the following tissues were separated (gloves were changed, and instruments were cleaned between the process for each different tissue to prevent cross-contamination between tissue samples): skin and fur (entire body) digestive contents (from stomach until the rectum) gastrointestinal tract (from oesophagus until the rectum) liver kidneys brain muscle sample (quadriceps) remaining carcass Tissue samples and blood samples were weighed and stored frozen (at approximately -20 °C) until extraction. Any unused samples will be discarded after the finalization of the report.EXTRACTION AND ANALYSIS Initially, a non-GLP analysis was made on the faeces samples, washing solvents and paper towels used for washing of two treated and one control rat. In the second step, the faeces samples from the remaining three treated animals were also analyzed in a non-GLP process. Study samples or subsamples of faeces were extracted in n-Heptane, and analysed at the Test Site by a validated GC-FID method (Test Site study code: FPBSTUDY-113 / CiToxLAB study code: 14/328-901AN, GLP; and Test site study code: FPBDOK-AN-318-01, non-GLP). According to the Sponsor’ request and based on the observed results of the study, additional analytical measurements were taken on designated frozen tissue samples of two test item treated animals (IDs: 2002 and 2003). In addition, necessary steps of the feasibility checking and/of cross-validation of the analytical method were also performed.The following tissues will be measured for both test item treated animals (IDs: 2002 and 2003): gastrointestinal tract (from oesophagus to the rectum) liver kidney brain
Statistics:
Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means and standard deviations.
Preliminary studies:
Preliminary results from dose range finding study not reported in the study.
Type:
absorption
Results:
There was no evidence of any systemic absorption of the test item.
Type:
distribution
Results:
No test item was detected in the internal organs examined
Type:
excretion
Results:
The only test item detected was in the faeces.
Details on absorption:
Additional analysis of gastrointestinal tract, liver, kidney and brain samples was performed for two test item treated animals. No test item was detected in those internal organs (
Details on distribution in tissues:
Additional analysis of gastrointestinal tract, liver, kidney and brain samples was performed for two test item treated animals. No test item was detected in those internal organs (
Details on excretion:
Analysis of the faeces showed the presence of the test item in the faeces at a range of 34.09% - 62.91% of the administered weight.
Metabolites identified:
not measured
Bioaccessibility testing results:
Bioaccessibility not analysed in this study.

RESULTS

MORTALITY/MORBIDITY

Single oral (gavage) administration of the test item at 2000 mg/kg bw did not cause any mortality.

 

CLINICAL SIGNS

Single oral (gavage) administration of the test item at 2000 mg/kg bw did not cause any test item related effects.

 

BODY AND TISSUE WEIGHTS

Single oral (gavage) administration of the test item at 2000 mg/kg bw did not cause any effects on the body weight or body weight gain of the animals.

One control animal (#1002) had high body weight loss on Day 3; the weight of digestive content as well as the liver weight of this animal was also extremely low. The data of this animal were not used for calculation.

 

TISSUE SAMPLE ANALYSIS

In the first round, analysis of faeces was performed. The measured weight of the oil in the faeces was within the range of 34.09% - 62.91% of the administered weight. No oil was found in the faeces of the control animal, and no oil was found in the dry or wet towels, or in the rinsing n-Heptane.

According to the Sponsor’ request and based on the observed results, additional analytical measurements were taken on designated frozen tissue samples of two test item treated animals (IDs: 2002 and 2003), where the lowest amount of test item was detected in the faeces (thus the highest possibility for detection of the test item in tissue samples). The tissues were selected as being the ones where the physicochemistry and physiological information indicated the highest possibility of detecting the test item if it had been absorbed systemically.

The following tissues were analyzed for both test item treated animals: gastrointestinal tract (from oesophagus until the rectum), liver, kidney and brain. No test item was detected in the internal organs (< LOQ, Limit of Quantification).

Results of analysis

Animal ID

Administered volume (mL)

Weight of administered volume (mg, input)

Sample type

Time of sampling

Weight of Oil in the sample (mg, output %)

1001

0.71

575.1

Dry towel

n/a

<QL

Wet towel

n/a

<QL

Rinsing heptane

n/a

<QL

Faeces

6 hours

<QL

Faeces

24 hours

<QL

Faeces

48 hours

<QL

Faeces

72 hours

<QL

SUM (mg) / % of input

0 / n.a.

2002

0.68

550.8

Dry towel

n/a

<QL

Wet towel

n/a

<QL

Rinsing heptane

n/a

<QL

Faeces

6 hours

71.6

Faeces

24 hours

127.0

Faeces

48 hours

23.4

Faeces

72 hours

<QL

SUM (mg) / % of input

221.9 / 40.29

2003

0.7

567

Dry towel

n/a

<QL

Wet towel

n/a

<QL

Rinsing heptane

n/a

<QL

Faeces

6 hours

74.6

Faeces

24 hours

118.7

Faeces

48 hours

<QL

Faeces

72 hours

<QL

SUM (mg) / % of input

193.3 / 34.09

2001

0.68

550.8

Faeces

6 hours

7.1

Faeces

24 hours

274.5

Faeces

48 hours

<QL

Faeces

72 hours

<QL

SUM (mg) / % of in put

281.6 / 51.13

2004

0.67

542.7

Faeces

6 hours

<QL

Faeces

24 hours

320.0

Faeces

48 hours

<QL

Faeces

72 hours

<QL

SUM (mg) / % of input

320.0 / 58.96

2005

0.68

550.8

Faeces

6 hours

<QL

Faeces

24 hours

330.2

Faeces

48 hours

16.3

Faeces

72 hours

<QL

SUM (mg) / % of input

346.5 / 62.91

n.a.: not applicable

QL: Quantification Limit

Note: Weight data were rounded to one decimal place, percentage values were rounded to two decimal places.

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
In conclusion, under the conditions of this study, single oral (gavage) administration of NovaSpec Base Oil at the dose level of 2000 mg/kg body weight did not cause any test item related effects. There was no evidence of any systemic absorption of the test item; the only test item detected was in the faeces.
Executive summary:

This study was performed to examine the absorption, distribution and excretion of NovaSpec Base Oil after a single oral (gavage) administration at the dose level of 2000 mg/kg body weight according to the following guidelines

-OECD Guidelines for Testing of Chemicals, No. 417 (2010)

-Directive 96/54/EC B.7 (OJL 248, 1996)

-Commission Regulation (EC) No. 440/2008, B.7.

 

Parameters monitored during the study included mortality/morbidity, clinical signs and food consumption daily. Urine and faeces was collected in metabolic cages for the following period from each animal:

0-6 hours post treatment

6-24 hours post treatment

24-48 hours post treatment

48-72 hours post treatment

 

Additionally, the metabolism bowl and all other relevant surfaces were washed with solvent (n-Heptane), wet and dry paper towels and the residual collected. Collected samples, residuals and paper wipers were stored frozen until extraction. After completion of the 72 hours collection period, all animals were sacrificed. At termination, animals were euthanised under pentobarbital anaesthesia by exsanguination. Animals were exsanguinated; blood samples for analytical purposes were taken by heart puncture into three tubes containing EDTA as anticoagulant. After exsanguination the following tissues were separated (gloves were changed, and instruments were cleaned between the process for each different tissue to prevent cross-contamination between tissue samples): skin and fur (entire body), digestive contents (from stomach until the rectum), gastrointestinal tract (from oesophagus until the rectum), liver, kidneys, brain, muscle sample (quadriceps), remaining carcass. Each tissue sample and blood sample was weighed and stored frozen until analysis.

 

Initially, a non-GLP analysis was made on the faeces samples, washing solvents and paper towels used for washing of two treated and one control rat. In the second step, the faeces samples from the remaining three treated animals were also analyzed in a non-GLP process. Study samples or subsamples of faeces were extracted in n-Heptane, and analysed at the Test Site by a validated GC-FID method.

 

Single oral administration of NovaSpec Base Oil at the dose level of 2000 mg/kg bw did not cause mortality and was not associated with any clinical signs.

 

Body weight and body weight gain was not affected by the treatment.

 

No effects were noted in food consumption.

 

At faeces analysis, approx. 34%-63% of the administered test item was found in the faeces. No test item was found in the rinsing solutions and paper towels used to remove residual test item from the metabolism bowls.

 

To provide more information, additional non-GLP analysis of gastrointestinal tract, liver, kidney and brain samples was performed for two test item treated animals. No test item was detected in those internal organs.

 

In conclusion, under the conditions of this study, single oral (gavage) administration of NovaSpec Base Oil at the dose level of 2000 mg/kg body weight did not cause any test item related effects. Analysis of the faeces showed the presence of the test item in the faeces at a range of approx. 34% - 63% of the administered weight. No test item was detected in the analyzed internal organs (gastrointestinal tract, liver, kidney and brain).

 

There was no evidence of any systemic absorption of the test item; the only test item detected was in the faeces. Recovery of the administered dose was 34%-63%. Recovery of this % amount is considered to be due to the analytical technique and is not indicative of absorption. 

Description of key information

Toxicokinetics, metabolism and distribution

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
5
Absorption rate - dermal (%):
1
Absorption rate - inhalation (%):
5

Additional information

Mineral hydrocarbons came under scrutiny late in the 1980s, largely as a result of new data from two 90-day feeding studies in Fischer-344 (F-344) rats. These studies showed that large doses of white mineral oils and waxes produced some inflammatory cell accumulation and pathology in livers (granulomas) of female F-344 rats, as well as in the mesenteric lymph nodes. These responses have been shown in various feeding studies to be strain- and species-dependent, and the F-344 rat responds to the greatest degree. Whilst the underlying mechanism(s) for strain and species-specific responses to MHCs remains unknown, the effects are not considered relevant to human clinical toxicity. However, as a result of these concerns, considerable efforts have been made to understand the subsequent fate of MHC’s following ingestion in the body. 

 

The results of the studies available indicate that accumulation is unlikely following exposure; and the data available indicates that few harmful effects are noted following ingestion. This is further confirmed via the evaluation of a two year carcinogenicity study utilising MHC’s. Within this study, whilst avariety of tumours developed in all groups, including the control group, all the neoplastic lesions were histologically similar to those known to occur spontaneously in rats, and no statistically significant increase in the incidence of any tumour type was found for either sex in the treated groups.. The authors of the study concluded that under the present experimental conditions, the high dose, about 2000-200,000 times higher than the current temporary acceptable daily intake (ADI), did not have any carcinogenic potential in rats. 

 

Evaluation of the available data and subsequent conclusions with respect to ADME are therefore as follows:

 

Absorption, Distribution and Excretion

 

A study to examine the Absorption, Distribution and Excretion of NovaSpec White Oils in Wistar Rats (Study Code: 14/265-048P) has been undertaken in accordance with:

·              OECD Guidelines for Testing of Chemicals, No. 417 (2010)

·              Directive 96/54/EC B.7 (OJL 248, 1996)

·              Commission Regulation (EC) No. 440/2008, B.7.

 

This study was performed to examine the absorption, distribution and excretion of NovaSpec White Oils after a single oral (gavage) administration at the dose level of 2000 mg/kg body weight according to the study design detailed below:

 

Dose group

Dose level

(mg/kg bw)

Dose volume

(mL/kg)

Control

0

2.5

Treated

2000

2.5#

#: Based on the relative density of the test item (0.81 g/mL as provided by the Sponsor).

 

The control animals were treated with distilled water.

 

Parameters monitored during the study included mortality/morbidity, clinical signs and food consumption daily. Urine and faeces was collected in metabolic cages for the following period from each animal:

 

·                   0-6 hours post treatment

·                   6-24 hours post treatment

·                   24-48 hours post treatment

·                   48-72 hours post treatment

 

Additionally, the metabolism bowl and all other relevant surfaces were washed with solvent (n-Heptane), wet and dry paper towels and the residual collected. Collected samples, residuals and paper wipers were stored frozenuntil extraction.After completion of the 72 hours collection period, all animals were sacrificed. At termination, animals were euthanised under pentobarbital anaesthesia by exsanguination.Animals were exsanguinated; blood samples for analytical purposes were taken by heart puncture into three tubes containing EDTA as anticoagulant. After exsanguination the following tissues were separated (gloves were changed, and instruments were cleaned between the process for each different tissue to prevent cross-contamination between tissue samples): skin and fur (entire body), digestive contents (from stomach until the rectum), gastrointestinal tract (from oesophagus until the rectum), liver, kidneys, brain, muscle sample (quadriceps), remaining carcass. Each tissue sample and blood sample was weighed and stored frozen until analysis.

 

Initially, a non-GLP analysis was made on the faeces samples, washing solvents and paper towels used for washing of two treated and one control rat. In the second step, the faeces samples from the remaining three treated animals were also analysed in a non-GLP process. Study samples or subsamples of faeces were extracted in n-Heptane, and analysed at the Test Site by a validated GC-FID method.

 

The results of this assessment are as follows:

 

1.      Single oral administration of NovaSpec White Oils at the dose level of 2000 mg/kg bw did not cause mortality and was not associated with any clinical signs.

 

2.      Body weight and body weight gain was not affected by the treatment.

 

3.      No effects were noted in food consumption.

 

4.      At faeces analysis, approx. 34%-63% of the administered test item was found in the faeces. No test item was found in the rinsing solutions and paper towels used to remove residual test item from the metabolism bowls.

 

5.      To provide more information, additional non-GLP analysis of gastrointestinal tract, liver, kidney and brain samples was performed for two test item treated animals. No test item was detected in those internal organs.

 

In conclusion, under the conditions of this study, single oral (gavage) administration of NovaSpec White Oils at the dose level of 2000 mg/kg body weight did not cause any test item related effects. Analysis of the faeces showed the presence of the test item in the faeces at a range of approx. 34% - 63% of the administered weight. No test item was detected in the analyzed internal organs (gastrointestinal tract, liver, kidney and brain). There was no evidence of any systemic absorption of the test item; the only test item detected was in the faeces. Recovery of the administered dose was 34%-63%. Recovery of this % amount is considered to be due to the analytical technique and is not indicative of absorption.

 

The results of oral acute toxicity study data along with significant evidence from the literature indicate that the substance is unlikely to be absorbed orally. In addition, there is sufficient evidence to indicate that significant absorption through the dermis would not occur on the basis of the available data. Inhalation exposure to the material in an acute toxicity test indicates that there are no effects; however the intrinsic properties indicate that exposure to vapours would not occur. Sufficient practical risk management measures are in place to avoid exposure to aerosols of the substance, and hence exposure via inhalation is predicted not to occur. Absorption rates are assigned on the following basis:

 

Oral: Almost all (95-99%) of ingested analogous food-grade mineral oil leaves the body unchanged in the faeces, 1-5% being absorbed as such by the intestinal mucosa. Studies in rats show that absorption of ingested n-alkanes is inversely related to molecular weight. The percentage retention of aliphatic hydrocarbons was inversely proportional to the number of carbon atoms and ranged from 60% for C14 to 5% C28 compounds. 5% is taken as a definitive value on the basis of the available literature, given that the test material was not present within any organs assessed in the absorption study.

 

Dermal: Sufficient literature data exists to demonstrate that that mineral oil does not effectively penetrate the skin beyond the stratum corneum, resulting in minimal (<1%) absorption of white mineral oils after topical exposure.

 

In the event that absorption does occur following exposure, NovaSpec Base Oil is likely to be metabolised to the associated fatty acid, followed by subsequent metabolic fate associated with fatty acids. However, significant absorption following exposure is not predicted to occur.

 

It is therefore considered that the substance is not absorbed when ingested.