Branched hexatriacontane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2011 - 11 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

The body weights for males 73 and 76 performed on the 15/07/11 appear to be incorrect
as they are not in line with body weight increase seen in the remaining males. In the
absence of any correlates to suggest a treatment affect, it was considered that these
animals were incorrectly weighed on the 08/07/11 in error. The data recorded for these
animals on the 08/07/11 will therefore be suppressed.
On the 25/10/11 the male 5hr observations were recorded approximately 1.5hrs late in
error.
On the 22/11/11 the residue for cage 100 was not recorded in error before the food was
topped up.
These are not considered to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetradec-1-ene, dimers, hydrogenated

Molecular formula:

C28H58

IUPAC Name:

Tetradec-1-ene, dimers, hydrogenated

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley Crl:CD (SD) IGS BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. The animals were acclimatised for at least fourteen days during which time their health status was assessed. A total of two hundred and twenty-four animals (one hundred and twelve males and one hundred and twelve females) were accepted into the study. At the start of treatment, the males weighed 223 to 301g and the females weighed 157 to 217g and were approximately eight weeks old.

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C, Teklad Global Certified Diet, Harlan Laboratories, UK, Ltd., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.

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 computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21±2ºC and 55 ±15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

The test item was prepared weekly and administered daily for 10 weeks by gavage, using a stainless steel cannula attached to a disposable plastic syringe, to three groups, each of twenty eight male and twenty-eight female F0 Generation Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twenty-eight male and twenty-eight female F0 Generation rats was dosed with vehicle alone (Arachis oil BP) 4ml/kg.

F1 generation: Selected F1 males and females (24 per dose group) were treated at the appropriate dose levels from Day 21 post partum for ten weeks during which they were evaluated for evidence of sexual maturation.
(The F2 generation were not treated, but killed after weaning.)

Details on mating procedure:

After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, for a maximum of 14 days, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum,
followed by the termination of all F0 male dose groups. The offspring selected for the F1 generation were dosed for at least 10 weeks and then paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving adults and their offspring were killed, followed by the termination of all F1 male dose groups.

Analytical verification of doses or concentrations:

yes

Remarks:

Verified within +/-10% of nominal concentration

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results are given in Appendix 27 and show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared weekly and stored at
approximately +4ºC under nitrogen.
Samples were taken of test item formulations and analysed for concentration of Hydrogenated oligomerisation product, including dimers and trimers, of tetradec-1-ene and alkene at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Samples were diluted in tetrahydrofuran to give a final theoretical conc approx 0.1mg/ml and analysed by GC against appropriate standard concentrations. The results indicate that the prepared formulations were within ±10% of the nominal concentration.

Duration of treatment / exposure:

10 weeks

Frequency of treatment:

daily

Details on study schedule:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP. Doses were prepared weekly, shown to be stable for at least 21 days. See below (study design) for further details of the study schedule.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

28 (F0) 24 (F1)

Control animals:

yes, concurrent vehicle

Details on study design:

Chronological Sequence of Study
i) Groups of twenty-eight F0 male and twenty-eight F0 female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
ii) A vaginal smear was prepared daily for twenty-one days prior to pairing for all F0 females.
iii) During Week 11 all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
v) Pregnant F0 females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight and clinical observations were also performed during this period.

vi) At Day 21 post partum, wherever possible, one male and one female offspring from the F0 mating phase were selected to form the next generation (F1). Surviving adult F0 females and unselected offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 females and selected organs were weighed for one unselected male offspring and one unselected female offspring.
vii) Following completion of the F0 female gestation and lactation phases, the F0 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
viii) Selected F1 males and F1 females were treated at the appropriate dose levels from Day 21 post partum for ten weeks during which they were evaluated for evidence of sexual maturation.
ix) A vaginal smear was prepared daily for twenty-one days prior to pairing for all F1 females.
x) Following ten weeks of treatment all F1 animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
xi) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
xii) Pregnant F1 females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight and clinical observations were also performed during this period.
xiii) At Day 21 post partum, all surviving F1 females and offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 females and selected organs were weighed for one male and one female offspring.
xiv) Following completion of the F1 female gestation and lactation phases, the F1 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.

Examinations

Parental animals: Observations and examinations:

Morbidity and mortality, clinical signs, body weight development, food and water consumption were monitored during the study.
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Oestrous cyclicity (parental animals):

Prior to pairing of females for the F0 and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The smears were examined microscopically and the stage of oestrous was recorded.

Sperm parameters (parental animals):

At necropsy of adult F0 and F1 males the following evaluations were performed:
i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture.
Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. Approximately 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) A sample of semen was preserved in formalin and then stained with eosin. A subsample was placed on a glass slide with a coverslip and a morphometric analysis of 200 sperm* was performed manually.

* = preserved in Bouins fluid then transferred in 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
v) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.
vi) For ii), iii), iv) and v) above samples from Groups 1 (Control) and 4 (high dose group) were evaluated.

Litter observations:

Litter Size, Sex Ratio and Viability.
Offspring Growth and Development: litter weights, offspring body Weight on Day 1, subsequent gain to weaning (Day 21 of age).
Sexual Maturation (F1 only).
Litter Observations. Clinical signs

Postmortem examinations (parental animals):

3.4.2 Pathology
Surviving adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 21 post partum. Surviving offspring (with the exception of offspring selected to form the F1 generation) were terminated via carbon dioxide asphyxiation. Following the termination of all adult females and offspring, all surviving males were killed by intravenous overdose of sodium pentobarbiturate agent followed by exsanguination.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium
polysulphide solution where applicable. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

3.4.2.1 Organ Weights
The following organs, removed from all F0 males and F0 females from each treatment
group that were killed at the end of the study, were dissected free from fat and weighed
before fixation:
Adrenals Pituitary (post fixation)
Brain Seminal vesicles (with coagulating gland and fluids)
Epididymides (total and caudal) Spleen
Kidneys
Testes
Liver
Thyroid/Parathyroid gland (post fixation)
Ovaries Uterus (with cervix and oviducts)
Prostate

The following organs were weighed from one male and one female offspring from the F0
and F1 pairings (where available):
Brain Spleen Thymus Uterus

3.4.2.2 Histopathology
The following tissues were preserved from all F0 males and females from each dose
group, in buffered 10% formalin except where stated:

Adrenals
Prostate
Right Epididymis *
Seminal vesicles (with coagulating gland)
Ovaries Uterus (with Oviducts and Cervix)
Right Testis* Vagina
Pituitary gland
Gross lesions
All tissues were despatched to the histology processing Test Site (Harlan Laboratories
Ltd, Zelgliweg 1, CH-4452 Itingen, Switzerland) (Principal Investigator: S Gäehler). The
tissues from all F0 and F1 control and high dose group animals, any animals dying during
the study and any animals which did not achieve a pregnancy were prepared as paraffin
blocks, sectioned at nominal thickness of 5m and stained with haematoxylin and eosin
for subsequent microscopic examination. In addition, sections of testes and
epididymides from all control and 1000 mg/kg bw/day males were also stained with
Periodic Acid-Schiff (PAS) stain and examined.

Microscopic examination was conducted by the Study Pathologist (Principal Investigator:Dr K Heider at AnaPath GmbH). A complete histopathology phase report, including methods is presented in Appendix 25.

Postmortem examinations (offspring):

The following organs were weighed from one male and one female offspring from the F0 and F1 pairings (where available):
Brain Spleen Thymus Uterus
Macroscopic abnormalities

Statistics:

Quantitative data was analysed to detect the significance of intergroup differences from control; statistical significance was achieved at p<0.05. The following parameters were analysed:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Absolute Organ Weights, Body Weight-Relative Organ Weights, Sperm Concentration and Sperm Homogeneity Resistance.

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module:

The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Transformed data were analysed to find the lowest treatment level with a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data was analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p>=0.05 (not significant)

Reproductive indices:

Live birth index (No of offspring alive on day 1 / No of offspring born * 100

Offspring viability indices:

Viability Index 1 (%) Number of offspring alive on Day 4/ Number of offspring alive on day 1 * 100
Viability Index 2 (%) Number of offspring alive on Day 7/ Number of offspring alive on Day 4 * 100
Viability Index 3 (%) Number of offspring alive on Day 14/ Number of offspring alive on Day 7 * 100
Viability Index 4 (%) Number of offspring alive on Day 21/ Number of offspring alive on Day 14 * 100
Viability Index 5 (%) Number of offspring alive on Day 21 / Number of offspring alive on day 1 * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected in test or control animals throughout the study period. The following clinical signs were considered unrelated to test
item toxicity:

The F0 male treated with 300 mg/kg bw/day, the F1 male treated with 1000 mg/kg bw/day, one of the F1 control females and the F1 female treated with 100 mg/kg bw/day that were found dead showed no clinical signs prior to death. The remaining F1 control female that was found dead on Day 32 had an increased respiratory rate, ptosis, lethargy and chromodacryorrhea on Day 31. The male treated with 300 mg/kg bw/day that was killed in extremis on Day 101 of the F0 generation had a mass, tiptoe gait, a red/brown stained snout, hunched posture and chromodacryorrhea prior to termination on Day 101. As previously discussed, microscopic examination revealed the deterioration of this animal was most likely to be due to an abscess formation in the right forelimb. The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 of the F0 generation showed a red/brown stained snout, noisy and laboured respiration, a decreased respiratory rate, lethargy, hunched posture and increased salivation prior to termination.

The control female that was killed in extremis on Day 97 of the F0 generation showed a red/brown stained snout, pilo-erection, lethargy and hunched posture on the day of termination. As previously discussed, microscopic examination revealed the deterioration of this animal was considered to be due to the development of thrombosis in the uterus after pregnancy. The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 of the F1 generation had a mass, laboured respiration, a decreased respiratory rate and lethargy on the day of termination. As previously discussed, microscopic examination revealed the cause of this death was most likely due to the abscess formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose. Incidents of red/brown staining of the external body surface, generalised fur loss, wound/scab formation, exophthalmos, chromodacryorrhea and noisy respiration were detected throughout the treatment period in treated and/or control animals from the F0 generation. Episodes of generalised fur loss and scab formation were also evident throughout the treatment period in treated animals from the F1 generation. These observations are commonly observed in laboratory maintained rats on long term studies and are considered of no toxicological significance. Isolated incidences of mass formation were noted in a few treated and control animals in both generations during the
treatment period. In the F0 generation these masses correlated either with lipoma in the thoracic region or mammary gland, decidual proliferation in uterus, abscess formation in the thoracic region or on the forelimb or adenocarcinoma of mammary gland. In the F1 generation these masses correlated either with adenoma in mammary gland, decidual proliferation in uterus, pleuritis or abscess formation on the forelimb. All these findings were considered to be incidental or possibly caused by a mal-dose and therefore are considered not to represent a toxic effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths considered to be related to test item throughout the study.

The following unscheduled deaths were seen during the treatment period:
One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F0
generation; however the aetiology of this death was not established. One male treated
with 300 mg/kg bw/day was killed in extremis on Day 101 of the F0 generation. The
deterioration of this animal was most likely to be due to an abscess formation in the right
forelimb. A further male from this treatment group was found dead on Day 53 of the F0
generation; however the aetiology of this death was not established. One control female
was killed in extremis on Day 97 of the F0 generation. The deterioration of this animal
was considered to be due to the development of thrombosis in the uterus after
pregnancy.

One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F1
generation. The cause of this death was most likely due to the abscess formation in the
right forelimb and the pleuritis which was possibly caused by a mal-dose. A further male
from this treatment group was found dead on Day 118 of the F1 generation; however the
aetiology of this death was not established. One control female was found dead on
Day 32 of the F1 generation. The cause of this death was considered to be pleuritis that
was possibly caused by a mal-dose. A further control female was found dead on Day 99
of the F1 generation. The cause of this death was possibly due to the marked decidual
proliferation with thrombosis in the uterus.

One female treated with 100 mg/kg bw/day was found dead on day 104 of the F1 generation; however the aetiology of this death was not established.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males from the F0 generation treated with 1000 and 300 mg/kg bw/day showed a
statistically significant increase in body weight gain during Week 7 of treatment. F0
generation males treated with 300 and 100 mg/kg bw/day also showed a statistically
significant increase in body weight gain during Week 15 of treatment. An increase in
body weight gain is considered not to represent an adverse effect of treatment and in the
absence of true dose related responses during Week 15 the intergroup differences were
considered not to be toxicologically significant. F0 generation males treated with
1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during
Week 15 of treatment. In isolation and in the absence of an effect on overall body weight
gain the intergroup difference was considered not to be of toxicological importance.

F0 generation females treated with 1000 and 300 mg/kg bw/day showed a statistically
significant increase in body weight gain during Week 3 of treatment. F0 generation
females treated with 100 mg/kg bw/day also showed a statistically significant increase in
body weight gain during Week 10 of treatment. An increase in body weight gain is
considered not to represent an adverse effect of treatment and in the absence of a true
dose related response during Week 10 the intergroup differences were considered not to
be toxicologically significant. F0 generation females treated with 100 mg/kg bw/day
showed a statistically significant reduction in body weight gain during Week 5 of
treatment. In the absence of a true dose related response the intergroup difference was
considered not to be toxicologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males:
No adverse effect on food consumption or food efficiency was detected in treated males
in either generation when compared to control animals.

Females:
Pre-pairing Food Consumptions: No adverse effect on food consumption or food
efficiency was detected in treated females in either generation during the maturation
phases.
Gestation Food Consumptions: No adverse effect on food consumption was detected in
treated females in either generation during the gestation phases.
Lactation Food Consumptions: No adverse effect on food consumption was detected in
treated females in either generation during the lactation phases.

Food efficiency:

no effects observed

Description (incidence and severity):

No adverse effect on food consumption or food efficiency was detected in treated animals in either generation.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No adverse effect on water consumption was detected.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect of treatment on sperm concentration, motility or morphology was apparent for males in either generation.
F0 males treated with 1000 mg/kg bw/day showed a statistically significant increase in the percent of sperm with normal morphology. An increase in this parameter is not considered to represent an adverse effect of treatment.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

From the F0 generation, one female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day was non-pregnant following positive evidence of mating, a further female from each of these treatment groups was also non-pregnant however both females did not show any positive evidence of mating and one female treated with 1000 mg/kg bw/day was non-pregnant following positive evidence of mating. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of the non-pregnancies.

Gestation: There were no differences in gestation lengths in either generation. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 24½ days.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinically observable signs of toxicity were detected in test or control animals
throughout the study period. The following clinical signs were considered unrelated to test
item toxicity:

The F1 male treated with 1000 mg/kg bw/day, one of the F1 control females and the F1 female treated with 100 mg/kg bw/day that were
found dead showed no clinical signs prior to death. The remaining F1 control female that
was found dead on Day 32 had an increased respiratory rate, ptosis, lethargy and
chromodacryorrhea on Day 31.

The male treated with 1000 mg/kg bw/day that was killed in extremis on
Day 106 of the F1 generation had a mass, laboured respiration, a decreased respiratory
rate and lethargy on the day of termination. As previously discussed, microscopic
examination revealed the cause of this death was most likely due to the abscess
formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose.
Episodes of generalised fur loss and scab formation were evident
throughout the treatment period in treated animals from the F1 generation. These
observations are commonly observed in laboratory maintained rats on long term studies
and are considered of no toxicological significance.

Isolated incidences of mass formation were noted in a few treated and control animals in both generations during the
treatment period. In the F1generation these masses correlated either with adenoma in mammary gland, decidual
proliferation in uterus, pleuritis or abscess formation on the forelimb. All these findings
were considered to be incidental or possibly caused by a mal-dose and therefore are
considered not to represent a toxic effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no deaths considered to be related to test item throughout the study. The
following unscheduled deaths were seen during the treatment period:

One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F1
generation. The cause of this death was most likely due to the abscess formation in the
right forelimb and the pleuritis which was possibly caused by a mal-dose. A further male
from this treatment group was found dead on Day 118 of the F1 generation; however the
aetiology of this death was not established. One control female was found dead on
Day 32 of the F1 generation. The cause of this death was considered to be pleuritis that
was possibly caused by a mal-dose. A further control female was found dead on Day 99
of the F1 generation. The cause of this death was possibly due to the marked decidual
proliferation with thrombosis in the uterus. One female treated with 100 mg/kg bw/day
was found dead on day 104 of the F1 generation; however the aetiology of this death was
not established.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation females treated with 1000 and 100 mg/kg bw/day showed a statistically
significant increase in body weight gain during Days 4 and 7 of lactation. The pattern of
body weight gain for the parent female during lactation is influenced by the increasing
demands of milk production from the growing litter. It is normally anticipated that females
in lactation will show body weight gain during the first two weeks of lactation, although
gain will lessen during the second week, and by the third week of lactation body weight
will remain static or loss is expected. The increased intergroup difference is therefore
considered not to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in the organ weights measured in
either generation.

For F1 males treated with 1000 mg/kg bw/day a statistically significant increase in kidney
weight both absolute and relative to terminal body weight was evident. In the absence of
a similar effect detected in F0 animals the intergroup difference was considered to be
incidental and unrelated to treatment.

F1 males treated with 100 mg/kg bw/day showed a statistically significant reduction in
pituitary weight both absolute and relative to terminal body weight. F1 females treated
with 1000 and 100 mg/kg bw/day also showed a statistically significant reduction in
pituitary weight both absolute and relative to terminal body weight whilst females treated
with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative
pituitary weight. In the absence of a true dose related response or in the absence of any
histopathological correlates the intergroup differences were considered not to be of
toxicological importance.
F1 males treated with 300 mg/kg bw/day showed a statistically significant increase in
thyroid weight both absolute and relative to terminal body weight. In the absence of a
true dose related response the intergroup difference was considered not to be of
toxicological importance.
F1 females treated with 1000 mg/kg bw/day showed a statistically significant reduction in
thyroid weight both absolute and relative to terminal body weight.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on
Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all
adhered to. The male from this treatment group that was found dead on Day 118 had a
mass under the right forelimb. The female treated with 100 mg/kg bw/day that was
found dead on Day 104 did not show any macroscopic abnormalities. One control
female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic
cavity and white fibrous adhesions on the lungs. The other control female that was
found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any
similar findings in terminal kill animals the intergroup differences were considered to be
incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One
female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a
malformed uterus. In the absence of true dose related reponses the intergroup
differences were considered not to be of toxicological importance. One female treated
with 100 mg/kg bw/day had a mass in the thoracic cavity and in the mammary tissue.
Microscopic examination revealed adenoma of the mammary gland and was considered
to be an incidental finding and of no toxicological importance.

Neuropathological findings:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects in either generation on female oestrous cycles or on the type or proportion of females with
anomalous oestrous cycles.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Sperm Analysis. There were no toxicologically significant effects on the concentration,
motility or morphology of samples of epididymal sperm. There were no treatment-related
effects on the concentration of homogenisation resistant epididymal or testicular
spermatid counts.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

From the F1 generation, one female treated with 300 mg/kg bw/day and one female
treated with 1000 mg/kg bw/day was non-pregnant following positive evidence of mating.
No histopathological correlates were evident in the male or female reproductive organs
to suggest the cause of the non-pregnancies. One female treated with 300 mg/kg
bw/day showed positive evidence of mating but failed to give birth to any live offspring.
Microscopic examination of this female showed a moderate unilateral pyelonephritis and
a marked pyometra in one uterine horn with surrounding peritonitis and adhesions to
duodenum and arteritis/periarteritis. These inflammatory processes were accompanied
by a marked reactive extramedullary hematopoiesis in the spleen. The male partner to
this female did not reveal any relevant findings, therefore the different inflammatory
processes in the female were considered to be the reason for the non-pregnancy. A
pseudopregnancy cannot however be totally excluded because in both the cervix and
vagina a mucification was noted.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The type, incidence and distribution of clinical observations observed for offspring in both
generations were consistent with that normally expected in offspring of the age examined
and did not indicate any adverse effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 Generation
Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on
Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all
adhered to. The male from this treatment group that was found dead on Day 118 had a
mass under the right forelimb. The female treated with 100 mg/kg bw/day that was
found dead on Day 104 did not show any macroscopic abnormalities. One control
female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic
cavity and white fibrous adhesions on the lungs. The other control female that was
found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any
similar findings in terminal kill animals the intergroup differences were considered to be
incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One
female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a
malformed uterus. In the absence of true dose related reponses the intergroup
differences were considered not to be of toxicological importance. One female treated
with 100 mg/kg bw/day had a mass in the thoracic cavity and in the mammary tissue.
Microscopic examination revealed adenoma of the mammary gland and was considered
to be an incidental finding and of no toxicological importance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
There were no toxicologically significant effects detected in body weight development in
either generation.

Females
Pre-pairing Body weights: There were no toxicologically significant effects detected in
body weight development in either generation during the maturation phases.

Gestation Body weights: There were no treatment related effects detected in body weight
development in either generation during the gestation phases.
Lactation Body weights: There were no toxicologically significant effects detected in body
weight development in either generation during the lactation phases.
F1 generation females treated with 1000 and 100 mg/kg bw/day showed a statistically
significant increase in body weight gain during Days 4 and 7 of lactation. The pattern of
body weight gain for the parent female during lactation is influenced by the increasing
demands of milk production from the growing litter. It is normally anticipated that females
in lactation will show body weight gain during the first two weeks of lactation, although
gain will lessen during the second week, and by the third week of lactation body weight
will remain static or loss is expected. The increased intergroup difference is therefore
considered not to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the attainment of sexual maturation.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For F1 males treated with 1000 mg/kg bw/day a statistically significant increase in kidney
weight both absolute and relative to terminal body weight was evident. In the absence of
a similar effect detected in F0 animals the intergroup difference was considered to be
incidental and unrelated to treatment.

F1 males treated with 100 mg/kg bw/day showed a statistically significant reduction in
pituitary weight both absolute and relative to terminal body weight. F1 females treated
with 1000 and 100 mg/kg bw/day also showed a statistically significant reduction in
pituitary weight both absolute and relative to terminal body weight whilst females treated
with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative
pituitary weight. In the absence of a true dose related response or in the absence of any
histopathological correlates the intergroup differences were considered not to be of
toxicological importance.

F1 males treated with 300 mg/kg bw/day showed a statistically significant increase in
thyroid weight both absolute and relative to terminal body weight. In the absence of a
true dose related response the intergroup difference was considered not to be of
toxicological importance.

F1 females treated with 1000 mg/kg bw/day showed a statistically significant reduction in
thyroid weight both absolute and relative to terminal body weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant macroscopic abnormalities detected in both the
F0 and F1 animals. The following macroscopic abnormalities were considered to be
unrelated to treatment with the test item:


Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on
Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all
adhered to. The male from this treatment group that was found dead on Day 118 had a
mass under the right forelimb. The female treated with 100 mg/kg bw/day that was
found dead on Day 104 did not show any macroscopic abnormalities. One control
female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic
cavity and white fibrous adhesions on the lungs. The other control female that was
found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any
similar findings in terminal kill animals the intergroup differences were considered to be
incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One
female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a
malformed uterus. In the absence of true dose related reponses the intergroup
differences were considered not to be of toxicological importance. One female treated

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic findings were within normal background range and not treatment-related.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

In total, for the F0 generation there were 27, 25, 25 and 27 females at 0 (control), 100,
300 and 1000 mg/kg bw/day respectively who gave birth to a live litter and successfully
reared young to weaning (Day 21 of age). From the F1 generation, there were 21, 23, 22
and 23 females at 0 (Control), 100, 300 and 1000 mg/kg bw/day respectively who gave
birth to a live litter and successfully reared young to weaning (Day 21 of age) and have
been included in the assessment of litter responses.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The type, incidence and distribution of clinical observations observed for offspring in both
generations were consistent with that normally expected in offspring of the age examined
and did not indicate any adverse effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

From the F1 generation, one control female showed total litter loss post partum. As there was no
consistency between the two generations and also no increase in mortality for offspring
for those litters surviving to weaning in either generation, these isolated occurrences
were considered to be incidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment on litter weights, offspring body weight on
Day 1 for either sex or subsequent body weight gain of each sex to weaning (Day 21 of
age) in either generation.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

F2 generation were sacrificed after weaning at day 21 therefore sexual maturation was not examined and F2 animals were not treated with the test item.

Applicant's summary and conclusion

Conclusions:

The oral administration of Hydrogenated oligomerisation product, including dimers and trimers, of tetradec-1-ene and alkene to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.

Executive summary:

The oral administration of Hydrogenated oligomerisation product, including dimers and trimers, of tetradec-1-ene and alkene to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.