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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9-Aug-2011 to 15-May-2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to established protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
No deviations were deemed to have affected the outcome of the integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes
EC Number:
810-258-3
Molecular formula:
not applicable; UVCB
IUPAC Name:
Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes
Test material form:
other: yellow liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Fifty two male and 52 female Sprague-Dawley rats (Crl:CD®(SD)) were received at Charles River on 09 Aug 2011 from Charles River Limited, Margate, Kent. On arrival, a sample of the males weighed 163-177 g and a sample of the females weighed 155-180 g. The animals were all approximately 6 weeks old. The animals were allowed to acclimate to the Charles River rodent toxicology accommodation for 13 days before the commencement of dosing. There was automatic control of temperature and humidity and target ranges were 19 to 23°C and 40 to 70%, respectively, with a minimum of 10 air changes per hour. Prior to animal arrival, procedures were changed from 15 air changes per hour as specified in the protocol, to 10 air changes per hour. As this change was approved by the company veterinarian and was a planned change to procedure, the deviation was considered not to have affected the outcome or integrity of the study.Daily monitoring indicated that temperature remained within the target ranges for the duration of the study. Humidity was outside the target ranges on 4 occasions (actual range 44-74%). The animals did not exhibit any adverse signs following these deviations from humidity and these were considered not to have any impact on the outcome or integrity of the study.A 12 hour light/dark cycle was in operation (light hours were 0700-1900 h).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethylcellulose (medium viscosity) (w/v), 0.1% TWEEN 80 in Water for Irrigation
Details on exposure:
Doses were administered orally by gavage at a dose volume of 10 mL per kg body weight, using a plastic gavage. The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from day 16 until parturition was complete, the dose volume was determined by the weight of the animal on day 16 of gestation.
Details on mating procedure:
A few days prior to the initiation of mating, the males were separated into individual grid bottom cages. Pairings were on a one male to one female basis. Animals were paired in numerical order within groups. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed. Vaginal lavages were taken daily early each morning from the day of
pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of a plug in situ or of sperm in such a lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of analyses of the dosing formulations prepared for use during Week 1 of the study were outside the ±10% acceptance criteria for Groups 2 and 3 (-33.6 and -69.4 % from nominal, respectively). The Group 4 formulations were within 1% difference from nominal. A re-run of samples confirmed the low result of the Group 2 formulations (-25% from nominal) but the Group 3 formulations were found to be within the acceptance criteria (0.8% from nominal). Because of these results, an additional sampling occasion for formulations prepared for use during Week 2 of the study was scheduled.The results of analyses of the dosing formulations prepared for use on Week 2 of the study were within the ±10% (-5.9 to 2.6%) from nominal indicating acceptable accuracy of formulation. The low coefficients of variation (<4%) indicated that these samples were homogenous.The results of the analysis of the dosing formulations prepared for use on Week 6 of the study were within ±10% (-2.4 and -3.8% for Group 2 and 3 formulations, respectively), however the Group 4 formulations were outside the criteria (-22.7%). A re-analysis of the Group 4 samples were then found to be +16% of nominal.On further review of the raw data, records indicate that the formulations prepared for use on Week 2 of the study were continually being stirred whilst being sampled and these results were all acceptable.The records for the formulations prepared during Week 1 and Week 6 of the study indicate that the formulations may not have been stirred whilst sampling took place and therefore this may account for the fact that results were not accurate and would also explain why the samples taken from the bottom of the container were a higher concentration than the samples taken from the top of the container (indicating they were not mixed appropriately during the sampling procedure).Formulations were stirred continuously throughout the dosing procedure and therefore the animals were considered to have been dosed correctly and the inaccurate results were considered to be due to sampling errors.
Duration of treatment / exposure:
The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating through to day 4 of lactation.Recovery animals were dosed as above and retained for a 14 day recovery period following completion of dosing.
Frequency of treatment:
The test and control items were administered by once daily oral gavage at dose levels of 0, 100, 500 and 1000 mg/kg/day for groups 1-4, respectively.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
10 animals per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were administered orally by gavage at a dose volume of 10 mL per kg body weight, using a plastic gavage. The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from day 16 until parturition was complete, the dose volume was determined by the weight of the animal on day 16 of gestation.The test and control items were administered by once daily oral gavage at dose levels of 0, 100, 500 and 1000 mg/kg/day for groups 1-4, respectively.The males were dosed for 4 weeks, starting 2 weeks prior to mating. The females were dosed 2 weeks prior to mating through to day 4 of lactation.Recovery animals were dosed as above and retained for a 14 day recovery period following completion of dosing. The recovery animals were necropsied and the organ weight and histopathology data were compared to those of the main study animals.

Examinations

Parental animals: Observations and examinations:
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which the first pups were born) was designated as Day 0 of lactation. The duration of gestation was calculated. Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.
Oestrous cyclicity (parental animals):
Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of a plug in situ or of sperm in such a lavage was designated Day 0 of gestation.
Litter observations:
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.
Where practicable, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any external abnormal decedent pup was preserved; externally normal ones were discarded.
Postmortem examinations (parental animals):
The main study males were killed when mating was completed and the animals had been dosed for at least 4 weeks. Recovery males were retained for a 14 day recovery period and then killed.The females were killed when convenient after all observations had been completed. Normally, both dam and litter were killed between Day 4 and Day 7 of lactation with the exception of the recovery females who were killed on or after Day 21 of lactation.Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.All main study and recovery animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system: all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded and the number of corpora lutea graviditatis on each ovary counted. Necropsy examinations were conducted by a trained technician and consisted of an external and internal examination and recording of observations for all animals. A veterinary pathologist was available for consultation during normal working hours.
Postmortem examinations (offspring):
Where practicable, animals found dead or killed prematurely were sexed, then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Surviving pups were also examined for externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, for optional further examination. Externally normal pups were discarded.
Statistics:
Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving test item. Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the Control group.Body weight, food consumption, haematology, coagulation, clinical chemistry and selected FOB and motor activity data were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis nonparametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, non-parametric equivalent of Student’s t test).Organ weight data was analysed as above and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.Incidence data was analysed as proportions in a Kruskal-Wallis analysis, or by categorical methods using contingency tables with the Fisher’s Exact Probability test or the Chi-squared test.
Reproductive indices:
The following indices of fertility were evaluated:
For each group:

Fertility Index (male) = Number siring a litter/Number paired

Fertility Index (female) = Number pregnant/Number paired

Gestation Index = Number bearing live pups/Number pregnant
Offspring viability indices:
For each litter and group:

Birth Index = Total number of pups born (live and dead)/Number of implantation scars

Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)

Viability Index = Number of pups live on Day 4 of lactation/Number live on Day 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 500 and 1000 mg/kg/day, there was a dose related increase in number of animals with salivation (12/20 animals at 500 mg/kg/day and 21/30 animals at 1000 mg/kg/day). Salivation was generally evident immediately after dosing and up to ca 2 hrs post dose.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 500 and 1000 mg/kg/day, microscopic findings were noted in the kidneys (basophilic tubules and hyaline droplets) of the main study males, considered to be treatment related.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Several pups in Dose Groups 3 and 4 displayed swelling or staining, respectively, on the ventral abdomen. Groups 1, 3 and 4 had pups that were cold to the touch.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day there was an increase in the number of animals giving birth to dead pups and also in the number losing more than 3 pups over Days 0-4 of lactation compared with Controls.
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
At 1000 mg/kg/day, there was an increase in the number of animals giving birth to dead pups and also in the number losing more than 3 pups over Days 0-4 of lactation, compared with Controls. Therefore, for the neonatal toxicity, the Lowest Observed Adverse Effect Level (LOAEL) was 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 500 mg/kg/day.From the findings on this study, the parental NOAEL was considered to be 100 mg/kg/day, however, findings at 500 and 1000 mg/kg/day were confined to salivation, and kidney findings which may be reversible and not relevant in man. The neonatal NOEL was considered to be 500 mg/kg/day.As findings in this study were considered not to be relevant in man, the parental NOAEL for man could be 1000 mg/kg/day.
Executive summary:

The objective of this study was to assess the toxicity of the test item in the rat after oral administration. The study was designed to assess possible effects on reproduction and/or development. The males were treated for 2 weeks prior to mating, through to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment). Recovery males and females had a 14 day recovery period prior to termination. The recovery animals were necropsied and the organ weight and histopathology data were compared to those of the main study animals.

The animals were monitored daily for any signs of ill health or reaction to treatment. Detailed functional observations were performed weekly, with additional functional observations performed on 5 males and 5 females per group on one occasion; week 4 for males and during early lactation for females.

Blood samples were also taken from 5 males and 5 females per group for laboratory investigations. Males were sampled during week 4 and females were sampled during lactation. In addition, histopathology was conducted on tissues from 5 males and 5 females

from Control and High dose groups.

Treatment with the registered substance was associated with salivation in animals treated at 500 and 1000 mg/kg/day. The salivation was generally evident immediately after dosing and was evident up to ca 2 hours post dose and may have been due to an unpleasant taste associated with the test item.

The type and distribution of neurotoxicity observations, the haematology parameters and the coagulation and clinical chemistry parameters did not indicate any association with treatment.

At 500 and 1000 mg/kg/day, there was a statistically significant increase in the kidney weights of the main study males, however the kidney weights of the recovery males (1000 mg/kg/day) were similar to those of Controls.

Histopathology findings in the male kidneys (basophilic tubules and hyaline droplets) were noted in main study males treated at 500 and 1000 mg/kg/day. There were associations between increased hyaline droplets in the proximal renal tubules, increased basophilic tubules and the increased kidney weights at these levels. The findings in recovery males were confined to 2/10 with basophilic tubules compared to 1 in Controls.

The hyaline droplets that form in the male rat renal tubules following the administration of materials such as the registered substance (volatile hydrocarbon) contain alpha-2microglobulin, and are unlikely to occur with hydrocarbon administration in man, as little or none of this protein is present in man, Greaves (2007).

Mating performance and pregnancy performance were similar across the groups.

At 1000 mg/kg/day, there was an increase in the number of animals giving birth to dead pups and also in the number losing more than 3 pups over Days 0-4 of lactation, compared with Controls. Therefore, for the neonatal toxicity, the Lowest Observed Adverse Effect Level (LOAEL) was 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 500 mg/kg/day.

From the findings on this study, the parental NOAEL was considered to be 100 mg/kg/day, however, findings at 500 and 1000 mg/kg/day were confined to salivation, and kidney findings which may be reversible and not relevant in man. The neonatal NOEL was considered to be 500 mg/kg/day.

As the parental findings in this study are thought not to be relevant in man, the parental NOAEL for man could be considered to be1000 mg/kg/day.

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