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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd September 2013 (animal preparation and dosing) to 16th September 2012 (necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
-
EC Number:
481-740-5
EC Name:
-
Cas Number:
848301-67-7
Molecular formula:
main general molecular formula: CnH(2n+2)
IUPAC Name:
C8-C26 branched and linear hydrocarbons – Distillates
Test material form:
other: liquid
Details on test material:
- Description: Clear colorless liquid
- Purity: 100%
- Date Received: 22 January 2013
- Storage Conditions: Room temperature in the dark
- Expiry Date: 12 October 2014

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were used. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 191 to 272g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan descried below in "Any other information on method".

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Corn Oil. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least twenty-five days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark.

Samples were taken of the test item formulations and were analyzed for concentration of 'Distillates (Fischer-Tropsch), C8-26 branched and linear'. The method used for analysis of formulations and the results obtained are given in the attached Appendix 12. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by gas chromtaography (GC) using an external standard technique. The test item have a chromatograohic profile consisting of multiple peaks.

Preparation of standard solutions
Syock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot of test item was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran to yield a solutin with a concentration of 1 mg/ml. The standard solutions contained the equivalent amount og vehicle to that of the relevant samples.

Analysis of samples
The formulations received were diluted with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran and then shaken to dissolve. Where necessary, sample solutions were further diluted with tetrahydrofuran to achieve the working concentration.

Preparation of accuracy samples
Samples of corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as per the test samples referred to in the previous paragraph.

Preparation of linearity standards
A range of standard solutions were prepared in tetrahydrofuran from a stock solution of 2.02 mg/ml by serial dilution covering the concentration range 0 to 2.02 mg/ml.

Instrumental Setup
GC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: ZB 1 HT (30 m x 0.25 mm id x 1 um film)
Oven temperature program: Ocen: 30°C for 0 minutes with 10°C/minute to 300°C for 5 minutes
Injection temperature: 250°C
Flame ionisation detector temperature: 250°C
Injectionn volume: 2 micor-l
Retention time: Profile of peaks from 5 to 25 minutes

Details on mating procedure:
Not described in study
Duration of treatment / exposure:
Day 5 to Day 19 of gestation,
Frequency of treatment:
Daily
Duration of test:
20 days - animals sacrificed on Day 20 after gestation
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
Post Mortem
All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/fetal and placental tissue visible

Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable fetus


Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p=0.05 (not significant)
Indices:
All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. Values given in appendices may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded valves therefore it is not always possible to calculate the exact group mean values from values presented in the appendices.

As the litter was the standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.


Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:

((Number of corpora lutea - number of implantations) / number of corpora lutea) x 100

Percentage post-implantation loss was calculated as:


((Number if implantations - number of live foetuses) / number of implantations) x 100


Sex Ratio

Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of male foetuses / number of females foetuses) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Adult Response

Mortality

There were no treatment-related deaths.

 

One control female was killedin extremison Day 15. Macroscopic observations revealed evidence of a physical trauma following the dosing procedure therefore this death was considered unrelated to test item toxicity.

 

 

 Clinical Observations

No toxicologically significant clinical signs were evident in treated females.

 

One female treated with 750 mg/kg bw/day showed an isolated incident of increased salivation on Day 6 and a further female from this treatment group had a decreased respiratory rate on Day 16. No such effects were detected in females treated with 200 or 50 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are considered not to be of toxicological significance.

 

One female treated with 200 mg/kg bw/day and one female treated with 50 mg/kg bw/day showed generalized fur loss between Days 6 and 7 and Days 16 and 20 (respectively). Observations of this nature are commonly observed in this type of study and in isolation are not considered to be related to test item toxicity. One female treated with 200 mg/kg bw/day had a damaged tail between days 13 and 20. This was a physical injury and unrelated to test item.

 

The female that was killedin extremison Day 15 showed red/brown staining around the snout, eyes and mouth, hunched posture, pilo-erection and respiratory pattern changes. One female treated with 750 mg/kg bw/day had a mass under the left forelimb. At necropsy, macroscopic observations for both females revealed evidence of a physical trauma following the dosing procedure therefore the clinical observations were considered unrelated to test item toxicity

Body Weight

No toxicologically significant effects in body weight development were detected.

 

Females treated with 750 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain between Days 5 and 6. The statistical significance was minimal and recovery was evident thereafter. Therefore in isolation and in the absence of an overall effect on body weight development the intergroup difference was considered of no toxicological significance.

 

Food Consumption

No adverse effect on dietary intake was detected.

 

Statistical analysis of the data did not reveal any significant intergroup differences.

 

 Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

 

Post Mortem Studies

No treatment-related macroscopic abnormalities were detected in treated females at terminal kill.

 

The female that was killed in extremis had a fluid filled thoracic cavity, a damaged trachea and an oil like substance in the stomach. One terminal kill female treated with 750 mg/kg bw/day had a mass under the left forelimb which was filled with a food like substance and a small hole in the trachea. These macroscopic observations were considered to be the result of a physical trauma following the dosing procedure and therefore unrelated to test item toxicity.

 

Litter Responses

Litter Data and Litter Placental and Fetal Weights

There was no treatment related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. There were also no toxicologically significant effects on pre-implantation losses or in sex ratio.

 

Females treated with 750 and 200 mg/kg bw/day showed a statistically significant reduction in pre-implantation loss. A true dose related response was not evident and a reduction in this parameter is considered not to represent an effect of treatment due to implantation occurring prior to the administration of the test item.

 

Fetal Examination

 For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of external, visceral or skeletal anomalies. The type of external, visceral and skeletal anomalies, were those commonly observed for this type of study. There were no findings that were considered to represent any known malformations.

 

During visceral assessment, litters from females treated with 50 mg/kg bw/day showed a statistically significant increase in the total percent of affected offspring. No significant increases were evident in any of the individual parameters examined and in the absence of a true dose related response the intergroup difference was considered not to be of toxicological significance. 

 

Applicant's summary and conclusion

Conclusions:
The oral administration of 'Distillates (Fischer-Tropsch), C8-26 branched and linear' to pregnant rats by oral gavage during gestation at dose levels of 50, 200 and 750 mg/kg bw/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 750 mg/kg bw/day.
No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item 'Distillates (Fischer-Tropsch), C8-26 branched and linear' on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study complied with the following guidelines:

 

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH)

 

 

Method

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 50, 200, and 750 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (corn oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

Results

- Mortality: There were no treatment-related deaths. One control female was killed in extremis on Day 15 due to a mal-dose.

- Clinical Observations: No clinically observable signs of toxicity were evident in treated females.

- Body Weight: No toxicologically significant effects in body weight development were detected.

- Food Consumption: No treatment related effects were detected in food consumption.

- Water Consumption: No adverse effect on water consumption was detected.

- Post Mortem Studies: No treatment related macroscopic abnormalities were detected.

- Litter Data and Litter Placental and Fetal Weights: No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

- Fetal Examination: No treatment-related effects were detected on external development or in the type and incidence of skeletal or visceral findings.

 

Conclusion

The oral administration of 'Distillates (Fischer-Tropsch), C8-26 branched and linear' to pregnant rats by oral gavage during gestation at dose levels of 50, 200 and 750 mg/kg bw/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 750 mg/kg bw/day.

No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day.