Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK Department of Health, 4 March 2009
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): NExBTL Renewable diesel
- Date received: 18 December 2007

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdRccHan:WIST
- Source: Harlan Laboratories UK Ltd
- Group mean weight at start of treatment: males 204-280 g; females 139-187 g
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12 (lights on 0600-1800 hr)

IN-LIFE DATES : Dose range finder
- Protocol signed 29 January 2008
- First treatment: 12 February 2008
- Necropsy: 4 March 2008


IN-LIFE DATES : Main study
- Protocol signed 29 January 2008; amendment/extension 28 July 2008
F0 GENERATION
- First treatment: 24 March 2008 (males) or 25 March 2008 (females)
- Necropsy: 29-31 July 2009 (males) or 4 July - 3 August 2008 (females)
F1 GENERATION
- First treatment: 31 July 2008 (males) or 1 August 2008 (females)
- Necropsy: 8-12 December 2008 (males) or 17 November - 16 December 2008 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Justification for vehicle used: the test substance was insufficiently soluble in water but readily soluble in arachis oil
- Concentration in vehicle: 0, 12.5, 62.5 and 250 mg/ml
- Dosing volume: 4 ml/kg bwt

Dosing volume adjusted based on most recent bodyweight.
Details on mating procedure:
F0 and F1 animals were paired (one male: one female) for up to 21 d. Females were examined and presence of sperm in a vaginal smear and/or presence of a vaginal plug was taken as positive evidence of mating. Pregnant females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples analysed by GC-FID. Results of stability and homogeneity studies showed that formulated dosing solutions were stable for at least 16 days and that the test substance was evenly distributed. Analyses for achieved concentration were within + or - 10% of nominal.
Duration of treatment / exposure:
At least 18 wk per generation, including pre-mating, mating, pregnancy and lactation.
Both sexes were treated for at least 11 wk prior to mating.
Frequency of treatment:
Daily
Details on study schedule:
F0 and F1 animals were mated after 11 weeks of treatment.
At weaning of the F1 offspring, groups of 24 male and 24 female offspring from each dose group were selected to form the F1 generation.
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250, 1000 mg/kg bwt/d
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
F0: 28
F1: 24
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection:
- based on results of dose range-finder investigation
- 1000 mg/kg bwt/d is a limit dose according to AnnexV to Directive 67/548/EEC
- treatments in excess of 1000 mg/kg bwt/d were considered unnecessary and undesirable

Pregnant females were observed at approx. 0830, 1230 and 1630 hr and around the period of expected parturition. The following information was retained:
- date of mating
- date and time of observed start of parturition
- date and time of observed completion of parturition
- duration of gestation

Twenty-four males and twenty-four females were selected at random from F0 litters from each dose group to form the F1 generation.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
Parental generations were monitored for clinical signs, bodyweight development and food and water consumption during the study.
Oestrous cyclicity (parental animals):
A vaginal smear was taken daily from F0 and F1 females for 3 wk prior to mating. The smears were allowed to dry, stained with diluted giemsa and examined microscopically. The stage of oestrous was recorded.
Sperm parameters (parental animals):
Sperm analysis was undertaken for F0 and F1 males from all dose groups:
- 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 limited to control and high dose groups (since no significant effects present)
Litter observations:
The following information was recorded for each litter:
- number of offspring born
- number of live offspring on day 1, 4, 7, 14, 21 post partum
- sex of offspring
- clinical condition of offspring from birth to weaning
- Individual offspring and litter weights on day 1, 4, 7, 14 and 21 post partum
- necropsy findings

F1 offspring were observed for sexual development:
- females: day of appearance of vaginal opening (separation of the labia)
- males: separation of the prepuce from the glans penis.
The bodyweight for each individual at the time of sexual maturation was noted.

The F1 offspring were observed for clinical signs from birth to weaning, including the occurrence of any gross external malformations.

Ano-genital distance was recorded for all F2 offspring on post partum day 1.
Postmortem examinations (parental animals):
Surviving adult females were sacrificed (sodium pentobarbitone/exsanguination) on post partum day 21. Surviving males were sacrificed following termination of all adult females and offspring. Necropsy examinations included:
- full gross examination (external, internal)
- following organs were weighed then fixed: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid gland, seminal vesicles (with coagulating gland), uterus (with cervix)

The corpora lutea of all ovaries and uterine implantation sites (ammonium polysulphide staining) were counted.

Selected tissues were from 10 F0 males and females from each dose group were subject to histopathological assessment (described in section 7.5.1).

Oocyte numbers from 10 selected control and high dose F1 females were determined microscopically (H&E staining) with oocytes classified as pre-antral phase, antral phase and pre-ovulatory phase
Postmortem examinations (offspring):
Pups were examined for the presence of gross internal and external malformations.
The following organs were weighed from 1 male and 1 female offspring from the F0 and F1 pairings: brain, spleen, thymus, uterus (females)
Statistics:
Organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights and offspring bodyweight data were assessed for dose response relationships by linear regression analysis, followed by ANOVA and Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were performed using Dunnett’s test otherwise the non-parametric Kruskal-Wallis ANOVA and Mann-Whitney U test were used. Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses. Histopathology data were analysed using Chi-squared (lesion incidence) or Kruskal-Wallis ANOVA.
Reproductive indices:
Reproductive indices included:
- stage of oestrus
- precoital interval
- fertility indices (mating index, pregnancy index)
- gestation length
- parturition index
- preimplantatyion loss
- post implantation loss
Offspring viability indices:
Offspring viability indices included:
- live birth index
- Viability on PND 4, 7, 14 and 21
- sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS
Increased salivation was present up to 1 hr after dosing for both F0 and F1 generation animals with the incidence increasing with treatment level. These findings are considered to be linked to the taste of the test substance formulations.

OESTRUS CYCLE ASSESSMENT
No treatment-related differences in oestrus cycle were detected between control and treated females from both the F0 or F1 generations, with females from all dose groups showing regular (4-5 d) cycles.

MATING, FERTILITY and GESTATION
No treatment-related differences in mating performance were detected for treated animals when compared controls from both the F0 and F1 generations, however the pregnancy index of the high dose group from the F0 generation was non-significantly lower compared to the controls. The study was therefore extended into a second generation to investigate this finding further, however no comparable reduction was apparent in the F1 generation

F0 pregnancy indices: 93%, 93%, 89%, 79%
F1 pregnancy indices: 91%, 96%, 96%, 96%
Results presented as (number pregnant/number mated x 100) for the control, low, intermediate and high dose groups, respectively. There were no statistically significant differences present.

The study report concluded that the results did not suggest a treatment-related effect on fertility/pregnancy.

There were no significant differences in length of gestation, which lasted between 22 to 23.5 d for both generations, regardless of treatment.

NECROPSY
A low incidence of gross macroscopic findings was found in all groups, including the controls, and considered unrelated to treatment with the test substance.

HISTOPATHOLOGY
Treatment-related histopathology was confined to the liver and kidneys:

LIVER:
Generalised hepatocyte enlargement was present in F0 females treated with 1000 mg/kg/day and 250 mg/kg/day however there was no convincing evidence of an effect in F0 males at any treatment level. A higher incidence of generalised hepatocyte enlargement was noted in high dose F1 females, with a marginal effect possibly present in high dose F1 males also. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is considered adaptive in nature.

KIDNEY:
Globular accumulations of eosinophilic material were observed in the tubular epithelium of F0 males treated with 1000 mg/kg/day (p<0.001) with isolated instances also seen at 250 mg/kg/day and 50 mg/kg/day and in one F0 male control. A higher incidence of globular accumulations of eosinophilic material was also noted in high and intermediate dose F1 males The condition was never graded as more severe than slight and there were no associated degenerative or necrotic tubular changes. The additional staining of kidneys from males with Mallory-Heidenhain stain indicated the presence of a2-microglobulin with the findings therefore considered not to represent an adverse effect of treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant systemic effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effect on reproduction

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

LITTER PARAMETERS
There was no statistically significant or treatment related effect on number of corpora lutea, implantation sites or number of live births in either generation although mean litter size for the intermediate and high dose groups from the P and F1 generations was approx. 8-10% lower than controls. This finding was considered not statsitically significant and considered of negligible toxicological significance.

OFFSPRING GROWTH AND DEVELOPMENT
Total litter weights were non-significantly decreased by 5-6% in high dose litter from both generations of the study however the finding was not statistically significant and considered of no biological relevance. Mean bodyweights were unremarkable.

There were no significant differences in onset of developmental and reflex milestones in litters from treated animals when compared to litters from controls over both generations.

There were no differences in age of sexual attainment for selected F1 male and female offspring when compared to controls, and these animals subsequently produced the second generation of the study.

There were no toxicologically significant differences in anogenital distances for F2 offspring when compared to concurrent controls.

No gross malformations were present.

NECROPSY
Macroscopic abnormalities were present at low incidence in all groups (including controls) and not considered to represent an effect of treatment.
No gross malformations attributable to treatment were present.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant systemic effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effect on reproduction
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effect on pup development

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effect on pup development

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No adverse effect on reproduction or fertility following gavage administration to male and female rats at dose levels up to 1000 mg/kg bwt/d over 2 generations. A NOAEL of 1000 mg/kg bwt/d was obtained for reproductive toxicity.
Executive summary:

The reproductive toxicity of NExBTL renewable diesel was examined in the rat following oral gavage administration over two consecutive generations at dose levels of 50, 250 and 1000 mg/kg/day. The study, which was GLP-compliant and included a sub-chronic reproductive toxicity phase, followed OECD guideline 416. Clinical signs were limited to salivation following dosing in the high and intermediate treatment groups which also showed increased water intake; both findings were considered secondary to the unpleasant taste of the test substance formulations.

Oestrus cycle, mating cycle and pre-coital intervals were comparable in control and parental animals although a non-significant increase in non-pregnant females was observed in the P-generation at 1000 mg/kg/d and 250 mg/kg/d. The study was therefore extended to include a second generation which showed a non-significant increase in fertility (all treated groups) when compared to controls. It was therefore concluded that the finding from the first generation was not indicative of an adverse effect on reproduction.

There was no statistically significant or treatment related effect on number of corpora lutea, implantation sites or number of live births in either generation although mean litter size for the intermediate and high dose groups from both P and F1 generations was approx. 8-10% lower than controls; this finding was not statistically significant and considered of negligible toxicological significance.

No gross malformations attributable to treatment were present in the off-spring. Ano-genital distance in F2 generation animals was unaffected by treatment.

Semen analysis for males from both generations and ovarian follicle development for F1 generation females were unremarkable.

Liver and kidney weights were elevated for high and intermediate males from both generations with treatment-related effects observed microscopically. Hepatic findings consisted of generalised hepatocyte enlargement for females treated with 1000 and 250 mg/kg/day from the P generation, and for animals of either sex treated with 1000 mg/kg/day from the F1 generation. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and considered to be adaptive in nature. Renal changes were characterised as globular accumulations of eosinophilic material in the tubular epithelium of males treated with 1000 and 250 mg/kg/day from both the P and F1 generations. This finding appeared consistent with alpha-2-microglobulin accumulation and was confirmed by the additional staining with Mallory-Heidenhain stain. Alpha-2-microglobulin is absent from humans and the findings were therefore viewed of negligible toxicological relevance.

It was concluded that oral administration of NExBTL renewable diesel to rats throughout the reproductive cycle over two generations at dose levels of up to 1000 mg/kg/day did not result in any toxicologically significant effects with an overall NOAEL of 1000 mg/kg bwt/d for reproductive and systemic toxicity.