Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9-Aug-2011 to 15-May-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed study according to accepted 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

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes

EC Number:

810-258-3

Molecular formula:

not applicable; UVCB

IUPAC Name:

Hydrocarbons, C12-C16, n-alkanes, isolkanes, 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 oral 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 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 4

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

Observations and examinations performed and frequency:

Mortality/Moribundity Checks
All animals were checked for viability early morning and as late as possible each day.

Detailed Clinical Observations
Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

Postdose Observations
All animals were examined for reaction to treatment daily during the course of the study. The onset, intensity and duration of any signs were recorded.

Body Weights
Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily in order to determine the correct dose volume to be administered. Body weights were recorded weekly during the recovery period (Days 7, 14 and 21 of lactation for recovery females).
For reporting, weekly weights have been presented for all males and for females prior to mating and then on Days 0, 7, 14, 16 and 20 of gestation and Days 1 and 4 of lactation.

Food Consumption
Food consumption was measured for both sexes weekly, starting 1 week before treatment and until pairing for mating.
After pairing, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation.
Additional food consumption was recorded for recovery females over Days 4-7 and 7-14 of lactation. This data is retained within the study data.

Ophthalmic Examinations
The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The following areas were evaluated: anterior, lenticular and fundic areas.

Detailed Functional Observations (Partial Examinations)
Once during the pre-treatment period (week -1) and weekly thereafter, a more detailed examination was made on all animals. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of body weight and food consumption data, and were performed at an approximately standardised time of day. Prior to the independent technician entering the animal room to perform the examinations, the cage card showing treatment group was removed from each cage, leaving the second pre-prepared card as the neurotoxicity animal identifier (so technician could not identify treatment groups of animals to be tested). In each cage, one or two animals had their tail marked to allow the technician to identify each animal.

Sacrifice and pathology:

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.

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.

Results and discussion

Results of examinations

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. There were a total of 2 premature decedent females on the study: Animal 64 (500 mg/kg/day) and Animal 71 (1000 mg/kg/day). These deaths were considered incidental for Animal 64 and not positively attributed to treatment for Animal 71.

Mortality:

mortality observed, treatment-related

Description (incidence):

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. There were a total of 2 premature decedent females on the study: Animal 64 (500 mg/kg/day) and Animal 71 (1000 mg/kg/day). These deaths were considered incidental for Animal 64 and not positively attributed to treatment for Animal 71.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 500 and 1000 mg/kg/day, occasional salivation noted in some males and females.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights statistically higher than Controls in main study males receiving 500 or 1000 mg/kg/day when adjusted for body wt.

Gross pathological findings:

no effects observed

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.

Histopathological findings: neoplastic:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical 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 HF-1000 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 HF-1000 (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.