Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A well conducted study to OECD guideline 416 with GLP. This robust study summary is prepared from the draft report.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Approximately eight weeks old.
- Weight at study initiation: At the start of treatment, the males weighed 229 to 301g and the females weighed 157 to 217g.
- Fasting period before study: None
- Housing: The animals were housed in a single air-conditioned room. Environmental conditions were continuously monitored.
- Diet ad libitum
- Water ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target values of 21±2ºC.
- Humidity (%): Target values 55 ±15% respectively.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12:12 Low intensity fluorescent light.

IN-LIFE DATES: From: June 2011 To: October 2011
Route of administration:
oral: gavage
Details on exposure:
Animals were treated daily at 0, 100, 300, 1000 mg/kg bw/day throughout the study (except for females during parturition where applicable). Doses were 4 ml/kg and arachis oil BP was used as control treatment.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
- Deviations from standard protocol: None
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test article in the formulations was determined by gas chromatography (GC) using an external standard technique.
Doses tested weekly were within the range 95 to 105% of nominal value throughout treatment.
Duration of treatment / exposure:
Groups of twenty-eight F0 male and twenty-eight F0 female animals were treated daily at the appropriate dose level (0, 100, 300, 1000 mg/kg bw/day) throughout the study (except for females during parturition where applicable). Doses were 4 ml/kg and arachis oil BP was used as control treatment.
Frequency of treatment:
Daily
Details on study schedule:
A vaginal smear was prepared daily for twenty-one days prior to pairing for all F0 females.
During Week 11 all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
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.
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).
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
28 Male; 24 female in both generations.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: historical toxicology data. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Positive control:
None.
Parental animals: Observations and examinations:
MORBIDITY/MORTALITY
Morbidity and mortality observations were performed twice daily during the week and once daily on weekends and public holidays.
CLINICAL OBSERVATIONS
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.
BODYWEIGHTS
Individual body weights were recorded for F0 males and F0 females on Day 1 (prior to treatment) and then weekly for F0 and F1 males until termination and weekly for F0 and F1 females until pairing. F0 and F1 females were weighed daily until mating was evident. Body weights for females showing evidence of mating were recorded on Days 0, 7, 14 and 21 post coitum. Females with live litters were weighed on Days 1, 4, 7, 14 and 21
post partum.
FOOD CONSUMPTION
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 - 7, 7 - 14 and 14 - 21 post coitum. For females with live litters, food consumption was recorded for the period covering Days 1 - 4, 4 - 7, 7 - 14, 14 - 21 post partum.
FOOD EFFICIENCY
Food efficiency (the ratio of body weight change / dietary intake) was calculated retrospectively for males for both the pre-mating and post-mating phases of the study. For females, food efficiency was only calculated for the pre-pairing phases of the study.
Due to offspring growth, milk production and weaning, food efficiency could not be accurately calculated for the gestation and lactation phases of the study.
WATER CONSUMPTION
Water intake was observed daily by visual inspection of water bottles for any overt change.
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):
The following characteristics were recorded for individual sperm analysis:

Concentration (millions/ml): Based on the total number of motile and non-motile cells identified.
Motility: The ratio of recorded motile cells to the total concentration.
Progressive Velocity (VSL): The straight line distance between the beginning and end of the track divided by time elapsed.
Litter observations:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 21 of age).
STANDARDISATION OF LITTERS
The offspring were selected randomly and where insufficient litters were available for offspring selection, additional offspring were selected
from remaining litters.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, semen assessments by histological examination of reproductive organs.

GROSS EXAMINATION OF DEAD PUPS: N/A
Postmortem examinations (parental animals):
Surviving adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 21 post partum. Following the termination of all adult females and offspring, all surviving males were then killed similarly. 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 including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
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.
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.

The tissues from all F0 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.

SEMEN ASSESSMENT
At necropsy of adult F0 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 preserved in Bouins fluid then transferred in 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
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.
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.
Postmortem examinations (offspring):
ORGAN WEIGHTS
The following organs were weighed from one male and one female offspring F1 pairings (where available):
Brain, Spleen, Thymus, Uterus.
HISTOPATHOLOGY
The tissues from 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.

SEMEN ASSESSMENT
At necropsy of adult 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 preserved in Bouins fluid then transferred in 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
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.
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.
OOCYTE NUMBER
Slides of the ovaries from ten control and ten high dose females from the F1 generation were prepared by serial sectioning of the tissue. Each slide was stained with haematoxylin and eosin. The sections of ovary were then examined microscopically.
Statistics:
Space limitation prevents full description. Quantitative data was subjected to statistical analysis; statistical significance was achieved at a level of p<0.05. Data were analysed using thedecision tree from the ProvantisTM Tables and Statistics Module. 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. Any transformed data were analysed to find the lowest treatment level that showed 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 was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysi is of variance (ANOVA). Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test which if significant, was followed by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test. Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses and offspring sex ratio) were analysed using non-parametric analyses.
Reproductive indices:
Standard reproductive indices determined were Pre-coital interval; Mating Index%; Pregnancy Index%; Gestation Index; Parturition Index; Implantation Losses (%); Number of corpora lutea; Number of implantation sites.
Offspring viability indices:
The following were calculated using guideline calculation methods:
Post implantation loss
Number of offspring born
Live Birth Index (%)
Number of offspring alive on Day 1 + Viability Index
Number of offspring alive on Day 4 + Viability Index
Number of offspring alive on Day 7 + Viability Index
Number of offspring alive on Day 14 + Viability Index
Number of offspring alive on Day 21 + Viability Index
Sex Ratio (% males) (Total number of offspring /Number of male offspring x 100.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths considered to be related to treatment amongst adult animals in either generation. No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in body weight development.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in body weight development.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related microscopic abnormalities were detected.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Analysis of formulation showed minimal (maximum 5%) deviation from nominal dose.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no treatment-related effects in either generation on oestrous cycles.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on the concentration, motility or morphology of samples of sperm from any source.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment reated effects on mating, fertility or gestation length in either generation.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Three males and one female were either killed in extremis or found dead with causes or mortality unrelated to treatment (see gross pathology below).
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. 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 was 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 thoracic region on in the forelimb or adenocarcinoma of mammary gland.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 a 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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 precent of sperm with normal morphology. An increase in this parameter is not considered to represent an adverse effect of treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating: There were no treatment-related effects on mating performance in either generation.
Fertility: There were no treatment-related effects on fertility in either generation.

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.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured in either generation.

For F0 males treated 1000 and 300 mg/kg bw/day a statistically significant reduction in adrenal weight both absolute and relative to terminal body weight was evident. In the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological importance. F0 animals of either sex from all treatment groups also showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 did not show any macroscopic abnormalities. The male treated with 300 mg/kg bw/day that was killed in extremis on Day 101 had enlarged adrenals, a pale liver and two masses near the right forelimb, each containing a brown viscous fluid. The male treated with 300 mg/kg bw/day that was found dead on Day 53 had fluid in the thoracic cavity. The control female that was killed in extremis on Day 97 did not show any macroscopic abnormalities. 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 with 1000 mg/kg bw/day had increased renal pelvic space in the right kidney. This observation is considered to be a congenital abnormalitiy and unrelated to treatment. One female treated with 300 mg/kg bw/day had a mass on the left flank. Microscopic examination revealed adenocarcinoma of the mammary gland and was considered to be an incidental finding and of no toxicological importance. A further female from this treatment group had a mass in the right kidney. In the absence of a similar effect seen in 1000 mg/kg bw/day animals the intergroup difference was considered not to be of toxicological importance. One control male had a mass in the right side of the thoracic cavity. A further control male had a mottled liver. One control female had a mass in the left side of the thoracic region, one control female had increased pelvic space in the right kidney and one control female had two masses in the right horn of the uterus. In the absence of treatment these findings were considered either a congenital abnormality or low incidence findings in rats of the strain employed on long term studies.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related microscopic findings were detected.

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects up to maximum tested dose.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths considered to be related to treatment or clinically observable signs of toxicity in test or control offspring animals.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no adverse effects of treatment on offspring viability
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in body weight development.
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no obvious adverse effects of treatment on the sexual maturation of offspring males or females.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute and body weight-relative organ weights for offspring did not indicate any adverse effects of treatment in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related abnormalities were detected at necrospy.
Histopathological findings:
no effects observed
Description (incidence and severity):
No treatment related microscopic abnormalities were detected
There were no toxicologically significant effects of treatment to detail in any of the following parameters, even at the highest dose of 1000 mg/kg bw/day:
VIABILITY (OFFSPRING)
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Females treated with 100 mg/kg bw/day from the F0 generation showed a statistically significant reduction in post implantation loss. The intergroup difference actually reflects a particularly good performance for the treated F0 females, and in the absence of a true dose related response is considered not to be of toxicological importance. Litter viability at Day 14 was increased in litters from all treated females in the F1 generation when compared to control litters. Overal litter viability in the F1 generation was also increased in litters from females treated with 1000 and 300 mg/kg bw/day. The intergroup differences actually reflect a particularly good performance for the treated F1 females, therefore is considered not to be of toxicological importance. Sex ratio (assessed as percentage males) at birth and subsequently to Day 21 of age was similar to control in all treatment groups over both generations, indicating no selective effect of treatment on survival for either sex. From the F0 generation, one female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day showed total litter loss post partum. 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.

CLINICAL SIGNS (OFFSPRING)
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.

BODY WEIGHT (OFFSPRING)
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.

SEXUAL MATURATION (OFFSPRING)
There were no treatment-related changes in the attainment of sexual maturation.

ORGAN WEIGHTS (OFFSPRING)
Assessment of intergroup differences in absolute and body weight relative brain, spleen, thymus and uterus weights for offspring did not reveal any obvious adverse effects of treatment in either generation.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment in either generation.

HISTOPATHOLOGY (OFFSPRING)
No treatment related microscopic findings were detected.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects up to maximum tested dose.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects up to maximum tested dose.
Reproductive effects observed:
not specified
Conclusions:
The No Observel Effect Level (NOEL) parental is 1000 mg/kg bw/day by oral gavage.
The No Observel Effect Level (NOEL) reproductiive toxicity (F0) is 1000 mg/kg bw/day by oral gavage.
The No Observel Effect Level (NOEL) reproductiive toxicity (F1) is 1000 mg/kg bw/day by oral gavage.
The No Observel Effect Level (NOEL) developmental toxicity (F0) is 1000 mg/kg bw/day by oral gavage.
The No Observel Effect Level (NOEL) developmental toxicity (F1) is 1000 mg/kg bw/day by oral gavage.


Executive summary:

In a two generation GLP study to the protocol OECD 416, 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 Adverse Effect Level’ (NOAEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Additional information

In a two generation GLP study to the protocol OECD 416, 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 Adverse Effect Level’ (NOAEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.

Supportive studies are available on similar substances:

The potential reproductive, developmental and subchronic toxicity of a C30 -C60 polyalphaolefin were evaluated in a dose range-finding study to aid in the selection of dosage levels for a definitive 91-day in utero study in rats. No test substance-related mortality or adverse clinical signs were observed in the F0 or F1 animals. There were no apparent test substance-related differences noted among the F0 or F1 study groups with respect to mean body weights, weight gains or gross necropsy findings. Similarly, no apparent test substance-related histopathological findings were observed in the liver, spleen or mesenteric lymph nodes of females from the F0 generation (1000 and 2000mg/kg/day groups) which were dosed for a total of 91 days. Mean F0 gestation length was similar between the groups and there were no signs of prolonged delivery or unusual nesting behaviours observed in any of the F0 females. F1 pup viability and external pup observations were comparable between the control, 100, 500, 1000 and 2000mg/kg/day groups throughout lactation. Although not statistically significant, the male-to-female pup ratio in the 2000mg/kg/day group was greater than the control group on lactation day 0.

The potential toxicity of the same substance was evaluated in a study following single daily oral doses by gavage for a minimum of 91 days. The animals utilized were the offspring of parental animals administered the test article. The in utero phase consisted of a control vehicle group and three doses of the test article suspended in polyethylene glycol 400 and administered at dosage levels of 100, 500 and 1000mg/kg/day. For the in utero phase, the F0 males were treated for a minimum of four weeks prior to mating. Treatment of the F0 females was initiated four weeks prior to mating and continued through lactation day 20. F0 females were allowed to deliver and rear their offspring. F1 pups were treated beginning at 22 days and continuing for the 91-day toxicity phase. No apparent toxicity was observed in F0 male and female rats including no effects on their fertility. In addition, F1 pups did not demonstrate any test article-related toxicity during the parturition and lactation phases. In the F1 rats during the 91 -day toxicity phase, minor gastrointestinal disturbances were seen in all groups, judged to be vehicle-related. No apparent test article-related clinical observations wre noted. There were transient changes in body weights, weight gain, food consumption, hematology parameters and organ weights at a few intervals, but were not considered to be biologically meaningful. A statistically significant increase in prothrombin time was seen in the males of the 1000mg/kg/day group, however, this change did not correlate with a decrease in platelets, gross necropsy findings or any lesions noted histopathologically. Therefore, this increase in prothrombin time was not considered to be biologically meaningful. There were no apparent gross necropsy observations or histopathologic lesions that could be related to treatment and no apparent toxic effects on the numerous parameters measured. Therefore, the NOEL was judged to be 1000mg/kg/day.


Short description of key information:
NOAEL=1000mg/kg - maximum tested dose.

Justification for selection of Effect on fertility via oral route:
Only two generation study available.

Effects on developmental toxicity

Description of key information
No treatment related adverse effects seen at maximum tested dose of 1000mg/kgbw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study to OECD Guidelines with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Approximately 10-12 weeks old - timed-mated day 3 gestation.
- Weight at study initiation: At the start of treatment females weighed 186 to 291g.
- Fasting period before study: None
- Housing: The animals were housed in a single air-conditioned room. Environmental conditions were continuously monitored.
- Diet ad libitum
- Water ad libitum
- Acclimation period: None
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target values of 21±2ºC.
- Humidity (%): Target values 55 ±15% respectively.
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12:12 Low intensity fluorescent light.

IN-LIFE DATES: From: 29 January 2012 To: 16 February 2012
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle dissolved test substance completely, was inert and formed stable solutions.
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test article in the formulations was determined by gas chromatography (GC) using an external standard technique. Doses tested weekly were within the range 95 to 105% of nominal value throughout treatment.

The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical
Services (Harlan Laboratories Ltd., Project No. 41101033); See 7.8.1.

Results show the formulations to be stable for at least twenty one days.

Formulations were therefore prepared once and stored at approximately +4°C in the dark. Samples were taken of each test item formulation and were analysed for concentration of Hydrogenated oligomerisation product, including dimers and trimers,of tetradec-1-ene and alkene at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within ± 3% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant.
Duration of treatment / exposure:
Between GD 5 and GD 19.
Frequency of treatment:
Daily.
Duration of test:
Test terminated on GD20.
Remarks:
Doses / Concentrations:
0, 100, 100, 1000mg/kgbw/day
Basis:
actual ingested
measured dose by gavage
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: historical toxicology data. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Maternal examinations:
CLINICAL OBSERVATIONS
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. An additional observation was also performed five hours after dosing during the normal working week. All observations were recorded.
BODYWEIGHT
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 Days 3, 5, 8, 11, 14, 17 and 20 of gestation.
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.
Ovaries and uterine content:
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) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/foetal and placental tissue visible
Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable foetuses were numbered according to their intrauterine position
Fetal examinations:
Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were
examined for skeletal development and anomalies. Following examination foetuses that were examined for skeletal development were placed in 100% glycerol.
Statistics:
The following parameters were analysed 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, an alternative multiple comparison test.

All caesarean necropsy parameters and foetal parameters: Kruskal-Wallis nonparametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Indices:
Pre- and post-implantation losses and sex ratio calculated by standard formulae.
Historical control data:
Not required - no observed effects to refer to.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS AND MORTALITY
There were no toxicologically significant clinical observations detected in any treated females. There were no unscheduled deaths.

BODY WEIGHT
No treatment-related effects in body weight development were detected.

FOOD CONSUMPTION
No adverse effects were detected in food consumption.

POST MORTEM
No toxicologically significant macroscopic abnormalities were detected in treated females.
One female treated with 1000 mg/kg bw/day had generalised fur loss at necropsy. This was an isolated incident and is considered not to be of toxicological significance. One female treated with 300 mg/kg bw/day had a mass in the left mammary gland. As similar observations were not apparent in animals treated with 1000 mg/kg bw/day, this was considered to be an isolated finding and is considered not to be of toxicological significance.

No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development.

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no adverse effect on in-utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and pre and post-implantation losses.
Animals treated with 300 mg/kg bw/day showed a statistically significant (p<0.01) increase in total corpora lutea when compared to control animals. In the absence of a true dose related response or any associated effects in the uterine parameters examined the intergroup difference is considered not to be of toxicological significance.
For all dose groups, there were no significant treatment-related trends in the proportion of foetuses (or litters) with evidence of visceral or skeletal anomalies. The type of visceral and skeletal anomalies were those commonly observed for this type of study.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The NOEL is considered to be 1000 mg/kg bw/day as no toxicologically significant changes were detected in the offspring parameters measured.
Executive summary:

In a guideline (OECD414) GLP study, oral administration of "Hydrogenated oligomerisation product, including dimers and trimers, of tetradec-1-ene and alkene" to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicologically significant effects at any dose level. The `No Observed Adverse Effect Level' (NOAEL) was therefore, considered to be 1000 mg/kg bw/day. No toxicologically significant changes were detected in the offspring parameters measured. The `No Observed Adverse Effect Level' (NOAEL) for reproductive and developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Additional information

In a guideline (OECD414) GLP study, oral administration of "Hydrogenated oligomerisation product, including dimers and trimers, of tetradec-1-ene and alkene" to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicologically significant effects at any dose level. The `No Observed Adverse Effect Level' (NOAEL) was therefore, considered to be 1000 mg/kg bw/day. No toxicologically significant changes were detected in the offspring parameters measured. The `No Observed Adverse Effect Level' (NOAEL) for reproductive and developmental toxicity was therefore considered to be 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Only study available.

Justification for classification or non-classification

There is no indication of toxicity to reproduction.