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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines (Ministry of Agroculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
-
EC Number:
482-220-0
EC Name:
-
Cas Number:
848301-69-9
IUPAC Name:
C18-C50 branched, cyclic and linear hydrocarbons – Distillates
Test material form:
other: Liquid
Details on test material:
Identification: GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear)
CAS No.: 848301-69-9
Description: Clear colorless liquid
Purity: No data available, the test item was regarded as 100% pure
Expiry Date (Retest Date): 09-May-2015
Storage Conditions: < 30 °C as given by the Sponsor, room temperature (15 - 25 °C), as handled at Harlan Laboratories, protected from light
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Number of Animals: 88 mated females, 22 mated females per group
Age (Day 0 Post Coitum): 11 weeks
Body Weight Range (Day 0 Post Coitum): 187 to 249 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually fol¬lowing room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.

Accommodation:
Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with pa-per enrichment (ISO-BLOX from Harlan Laborato-ries B.V., Netherlands).

Diet:
Pelleted standard Harlan Teklad 2018C (batch no. 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.

Water:
Community tap-water from Füllinsdorf and Itingen was available ad libitum in water bottles.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a solution was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Dose formulations were stored in the refrigerator at 5 ± 3 °C in dark glass beakers.

Vehicle
Identification: Corn oil
Source: Carl Roth GmbH
Batch Number: 103197718
Expiry Date: 27-May-2014
Storage Conditions Room temperature (15 - 25 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (8 days in the refrigerator at 5 ± 3 °C protected from light). Towards the end of the study, samples were taken from the middle to confirm concentra-tion. The aliquots for analysis of dose formulations were delivered at ambient temperature to Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland and stored there at -20 ± 5 °C until analysis.

The samples were analyzed with a GC-system equipped with a FID. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were not discarded without written consent from the study director.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum. Mating was performed in Füllinsdorf. After successful mating the animals were delivered on day 0 post coitum to Itingen to perform the rest of the study.

Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
15 days
Frequency of treatment:
Daily
Duration of test:
Day 6 - 20 post coitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
The group identification and animal numbers assigned to treatment are stated in the following table:

Allocation and Group 1 Group 2 Group 3 Group 4
Dose Levels Control
mg/kg bw/day 0 50 200 1000

Females 1-22 23-44 45-66 67-88



Examinations

Maternal examinations:
The following observations were recorded:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: Recorded at 3-day intervals: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.
Body Weights: Recorded daily from day 0 until day 21 post coitum.

Pathology
At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:

Microdissection Technique

For Microdissection technique (sectioning/dissection technique) [see References (2)] at least one half of the fetuses from each litter were fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This includes detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

Skeletal Examination
The remaining fetuses were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The specimens were preserved individually in plastic vials. The foetuses were sent to the principal investigator and evaluated by Harlan Laboratories Ltd. The principal investigator was notified prior to shipment. After the evaluation the fetuses were send back to Harlan Laboratories Ltd, 4452 Itingen / Switzerland and will be archived there.

Investigations were performed by personnel appointed by the principal investigator and were conducted according to Harlan Laboratories Ltd., standard operating procedure 189.15 “Evaluation of Fetal Skeletons”. Fetuses were examined under a binocular microscope. Fetuses were examined in mixed group order. Each litter was examined in sequential order.

The assessment included, but not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles.

All findings were recorded on the appropriate proforma. These findings were collated and tabulated. These tables were audited by Harlan Laboratories Ltd., quality assurance department and dispatched together with the raw data to the study director.

Fetuses with abnormalities were photographed, when considered appropriate.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, macroscopical findings, reproduction and skeletal examination data:

• Means and standard deviations of various data were calculated and included in the report.

• The Dunnett-test [see References (4)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (5)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (6)] was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Viability / Mortality
All females survived until the scheduled necropsy.

Clinical Signs
No test item-related clinical symptoms or signs were observed at any dose during the study.

Incidentally towards the end of the study, one female at 200 mg/kg bw/day showed a cloudy inclusion in the right eye.

Food Consumption
No test item-related changes were observed in the food consumption during the study.

Body Weights
Mean absolute body weight and body weight gain were not affected by treatment with the test item.

Corrected body weight gain (corrected for the gravid uterus weight) was not affected by treat-ment with the test item (+10.9%, +10.3%, +11.3%, +11.5% in order of ascending dose level).

Reproduction Data
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item.

Pathology (Maternal Data)
Macroscopic Findings
No findings were observed at any dose level during macroscopical examination.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Observations (Fetal Data)

External Abnormalities and Variations
No test item-related findings were observed during external examination of the fetuses in any group.

At 200 mg/kg bw/day, one pup had incidentally a short lower jaw (confirmed by fetal pathology).

Sex Ratios
No test item-related effects on the sex ratio of the fetuses were noted in any group.

The proportion of male fetuses was 50.0%, 45.8%, 50.7% and 52.1% in order of ascending dose level.

Body Weights
No test item-related effects on fetal body weights were noted.

Mean body weights of live fetuses calculated on litter and individual basis were statistically significantly increased at 1000 mg/kg bw/day (+6% when calculated on a litter basis for male and female fetuses).

Visceral Abnormalities and Variations
No test item-related findings were noted.

At 50 mg/kg bw/day, one fetus had a severely dilated renal pelvis. At 200 mg/kg bw/day, one fetus had a malpositioned subclavian artery and at 1000 mg/kg bw/day, one fetus showed a situs inversus. All these isolated abnormalities were considered to be incidental.

The type and frequency of commonly noted variations were similar in nature for the groups treated with the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related.

Bone Abnormalities and Variations
No test item-related findings were noted.

At 50 mg/kg bw/day and 200 mg/kg bw/day, there were two fetuses that showed similar find¬ings. One fetus had fused mandibles and lower incisors, misshapen exoccipital/basisphenoid and absent/reduced in size cervical vertebral arches. Another fetus showed a short lower jaw, fused mandibles, lower incisors and misshapen basisphenoid. However, in isolation and in the absence of any dose treatment relationship, this is not considered to be an adverse effect of treatment on fetal development.

The type and frequency of commonly noted variations were similar in nature for the groups treated with the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Analysis of Dose Formulations

The GTL Base oil distillate peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no significant peak appeared at the retention time of GTL Base oil distillate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.

 

The GTL Base oil distillate concentrations in the dose formulations ranged from 82.6% to 119.5% with reference to the nominaland were within the accepted range of±20%,with exception of sample of group 2-top prepared on 17-Aug-2013 (71.1%).However, the result was acceptedas homogeneity was in range.

 

The homogeneous distribution of GTL Base oil distillate in the preparations was approved because single results found did not deviate more than 13.6% from the corresponding mean and met the specified acceptance criterion of ≤15%.

 

In addition, the test item was found to be stable in application formulations when kept eight days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

Applicant's summary and conclusion

Conclusions:
This study is an investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) to rats. The test item was administered in corn oil as vehicle at dose levels of 50, 200 and 1000 mg/kg body weight/day.

No maternal toxicity was observed at any dose level.

The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item and also no effects on the development of fetuses were observed at any dose level. However, the fetal body weight was slightly statistically significantly increased at 1000 mg/kg bw/day (5.1 ± 0.2 g for the high dose-group versus 4.9 ± 0.2 g for the controls and 5.0 ± 0.2 g and 4.9 ± 0.3 g for the low and mid dose-groups, respectively). A test item-related effect cannot be excluded, most probably due to the higher nutritional value that the test item provides.

Based on this result a maternal NOEL (No Observed Effect Level) for GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear), was considered to be 1000 mg/kg body weight/day and a fetal NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/ day.

Under the conditions described for this study, GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) did not reveal teratogenic potential up to and including a dose of 1000 mg/kg body weight/day.
Executive summary:

Purpose

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6post coitum(implantation) to day 20 post coitum (the day prior to Caesarean section).

 

Rationale for Choice of Species, Route of Administration and Dose Levels

 

The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.

 

Based on the results of a OECD408 study (Harlan Study Number2041/0061) the dose levels of 0, 50, 200, and 1000 mg/kg bw per day were selected for the present study

  Guidelines / Regulations

This study was performed in accordance with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     OECD guidelines (OECD guideline for testing of chemicals proposal for updating guideline 414, prenatal developmental toxicity study. Adopted: 22nd January 2001) and EEC guidelines (EC 2004/73 B31, dated April 29, 2004).

 

·     US-EPA guidelines (Health Effects Test Guidelines, OPPTS 870.3700 ‘Prenatal Development Toxicity Study’, EPA 712-C-98 -207, dated August 1998).

 

·     Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).

Maternal Data

Mortality / Viability

All females survived until the scheduled necropsy.

 

Clinical Signs

No test item-related clinical symptoms or signs were observed at any dose during the study.

 

Food Consumption

No test item-related changes were observed in the food consumption during the study.

 

Body Weights

Mean absolute body weight, body weight gain and corrected body weight gain were not affected by treatment with the test item.

 

Reproduction Data

The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item.

 

Macroscopic Findings

No findings were observed at any dose level during macroscopical examination.

 

Fetal Data

External Abnormalities and Variations

During the external examination of the fetuses, no test item-related findings were observed.

 

Sex Ratios

No test item-related effects on the sex ratio of the fetuses were noted in any group.

 

Body Weights

Mean body weights of live fetuses were slightly increased at 1000 mg/kg bw/day. The minor increase was not in the range of the historical control data and an effect of the test item cannot be excluded.

 

Visceral Abnormalities and Variations

No test item-related findings were noted during external and fresh visceral examinations of fetuses.

Bone Abnormalities and Variations

No test item-related findings were noted during skeletal examination of the fetuses. No delay in the stage of ossification was observed.

The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item and also no effects on the development of fetuses were observed at any dose level. However, the fetal body weight was slightly statistically significantly increased at 1000 mg/kg bw/day (5.1±0.2 g for the high dose-group versus 4.9±0.2 g for the controls and 5.0±0.2 g and 4.9±0.3 g for the low and mid dose-groups, respectively). A test item-related effect cannot be excluded, most probably due to the higher nutritional value that the test item provides.

 

Based on this result a maternal NOEL (No Observed Effect Level) for GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear), was considered to be 1000 mg/kg body weight/day and a fetal NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/ day.

 

Under the conditions described for this study, GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) did not reveal teratogenic potential up to and including a dose of 1000 mg/kg body weight/day.