Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No biologically or toxicologically relevant effects on fertility or reproduction in a GLP-compliant guideline study up to an oral limit dose of 1000 mg/Kg bw/day.

Reproductive Toxicity NOAEL (Rat) = 1000 mg/Kg bw/day

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health, 4 March 2009
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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)
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.
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
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
Key result
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
Key result
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
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
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.
Key result
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
Key result
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
Key result
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
Key result
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
Reproductive effects observed:
not specified
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.

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
Quality of whole database:
Only reproductive toxicity study available for assessment
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a key study (Harlan Laboratories Ltd., 2009), 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 considered of negligible toxicological significance. 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 bw/d for reproductive and systemic toxicity.

Effects on developmental toxicity

Description of key information

No biologically or toxicologically relevant effects on development in two OECD Guideline 414 studies (rats and rabbits) when tested at doses up to 1000 mg/Kg bw/d.

Developmental Toxicity NOAEL (Rat and Rabbit): 1000mg/Kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-02-25 to 2020-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations were considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations were considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations were considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Neste Oyj (Keilaranta 21, Espoo, PL95, 00095 Neste, Finland); Batch (Lot) Number: K31/NEXBTL-32
- Expiration date of the lot/batch: 2024-06-12
- Purity test date: 2019-06-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: Considered stable for 6 hours at ambient temperature
- Stability under test conditions: Considered stable for 6 hours at ambient temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stable for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C)


FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear colourless liquid

OTHER SPECIFICS
- Specific gravity / density: 780.2 kg/m3 at 15°C
- Stability in vehicle: Arachis oil - Stability for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C), has been confirmed over the concentration range 50 to 700 mg/mL (suspensions), project 20223624.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland (Sulzfeld, Germany)
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 181 - 263 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: On arrival and following randomization, females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 52 to 54%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-02-18 and 2020-02-20 To: 2020-06-18
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized by stirring to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator.

Test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 50, 150, and 500 mg/mL for the control, low-, mid-, and high-dose groups, respectively.
- Amount of vehicle (if gavage): 2 mL/Kg
- Lot/batch no. (if required): Not specified (Source: Fagron (Capelle aan de IJssel, The Netherlands))
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20223624).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10% for solutions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20223624) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20223624.
Details on mating procedure:
Time-mated female Wistar Han Rats (Crl:WI(Han)) were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
From Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
Once daily oral gavage 7 days a week
Duration of test:
Day 6 to Day 20 post-coitum, inclusive
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control (Arachis Oil - Group 1)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Low Dose (Group 2)
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid Dose (Group 3)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High Dose (Group 4)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.

The dose levels were selected based on the results of the range finder in the rat (Test Facility Reference No. 20223626) in which 1000 mg/Kg/day was tested. No maternal or developmental toxicity was observed and 0, 100, 300 and 1000 mg/Kg/day were selected as dose levels for the main study. The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Rationale for animal assignment (if not random): At arrival, animals were randomly assigned to groups
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and
lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. Cage debris was examined to detect premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-
21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post-coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The uterus and thyroid gland were weighed.

OTHER:
- Clinical Pathology: Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected, between 7:00 and 9:00 a.m., from the jugular vein in the animal facility.

Thyroid Hormone (Immulite)
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for the parameters listed below:

1) Triiodothyronine (T3)
2) Thyroxine (T4)
3) Thyroid-Stimulating Hormone (TSH)

The values for T3 levels generated using the Immulite were not reported, but kept in study data. Part of the remaining volume was used for measurement of T3 using LC-MS.

Hormone Analysis Triiodothyronine (T3) (LC-MS)
Remaining samples of the Immulite thyroid hormone analysis were analyzed for T3 according to the bioanalytical method validated in Test Facility Study No. 20213516. After receipt, the samples were stored in an ultra-low freezer (≤ -75°C) until analysis.

- Organ Weights – F0-Generation: The thyroid gland and uterus were weighed at necropsy for all animals. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) were calculated.

- Histology - F0-Generation
Thyroid glands of all animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The sex of each fetus based on the anogenital distance (\not for animals found dead, sacrificed before planned necropsy, or that delivered).

In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. Litters of females surviving to scheduled necropsy were subjected to detailed external, visceral and skeletal examinations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

- External examinations: Yes: [all per litter]
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. For late resorptions, a gross external examination was performed.

- Soft tissue examinations: Yes: [half per litter]
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1972).

- Skeletal examinations: Yes: [half per litter]
All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in potassium hydroxide. Thereafter, they were stained with Alizarin Red S by a method similar to that described by Dawson (1926).

Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). As skeletal malformations were suspected for fetus (A014-10), selected for visceral examination, this fetus was also subjected to skeletal examination.

All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

- Head examinations: Yes: [half per litter]
The heads were removed from one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique (1965).
Statistics:
For information on statistics, please see section 'Any other information on materials and methods, incl. tables'.
Indices:
Maternal Variables
1) Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.

2) Corrected Body Weight Gains: Body weight determined on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.

3) Relative Food Consumption: Calculated against the body weight for scheduled intervals

4) Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum

Reproduction and Developmental Variables
1) Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) x 100

2) Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / (number of implantation sites)) x 100

3) Viable fetuses affected/litter (%): (number of viable fetuses affected/litter / number of viable fetuses/ litter) x 100
Historical control data:
Historical Control Data is presented in Appendix 3 of the final study report.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Salivation observed after dosing, which showed a dose-related incidence trend, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity, likely related to the taste of the test material.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test imaterial.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed through the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level over the study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total triiodothyronine (T3) were considered unaffected by treatment.

2 females in the control group (Nos. 9 and 17) had serum levels of thyroid stimulating hormone (TSH) well above the upper limit of the historical control range. Consequently, decreased TSH serum levels were noted in all treatment groups, (0.66x, 0.71x and 0.61x of controls for the 100, 300 and 1000 mg/Kg/day groups, respectively). The difference was not statistically significant and all mean values remained within the normal range. There was no dose-response relationship as well and therefore, this finding was not considered to be treatment-related.

At 1000 mg/Kg/day, serum levels of total thyroxine (Total T4) were slightly decreased (0.90x of controls). The effect was considered minimal and no statistical significance was reached. As the mean values remained well within the historical control range, this was also not considered to be treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related thyroid gland weight changes were observed through the study period.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings in the thyroid glands.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, two females at 1000 mg/Kg/day were not pregnant (Nos. 73 and 87). All other females were pregnant with viable fetuses. Given that treatment in this type of study starts after implantation, these cases of non-pregnancy were considered incidental and unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal weights (male, female and combined) were comparable to the controls for all treatment groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Male:female ratio was unaffected by treatment up to 1000 mg/Kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed on litter size of any group.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of fetuses (litters) available for morphological examination were 240 (22), 232 (22), 233 (22) and 207 (20) in the control, 100, 300 and 1000 mg/Kg/day groups, respectively.

No treatment-related effects on external morphology were observed following treatment up to 1000 mg/Kg/day.

Two fetuses were externally malformed. One fetus (A078-08) in the 1000 mg/Kg bw/day dose group had a small lower jaw and no eye bulges, and one control group fetus (A014-10) missed a digit. Skeletal findings substantiated these malformations, but as they occurred in a control fetus, singly and/or occurred previously in historical control fetuses, they were considered chance findings. No external variations were observed in any dose group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/Kg/day.

Skeletal malformations occurred in 2 (2), 2 (2), 0 (0) and 3 (3) fetuses (litters) in the control, 100, 300 and 100 mg/kg/day groups, respectively.

At the high dose, one fetus (A078-08) exhibited a small lower jaw, missing eye bulges, and its underlying anomalies also had a vertebral anomaly. This latter malformation also occurred in the control fetus with ectrodactyly (A014-10) and in a 100 mg/Kg bw/day dose group fetus (A027-10) (together a with small orbit). The single occurrence and group distribution of this anomaly does not indicate a relationship with treatment with the test material and therefore was considered a chance
finding.

Other malformations observed at 1000 mg/Kg bw/day were sternoschisis (fetus A076-08) and bent limb bones (fetus A084-04). Both these findings were also observed in control and/or the 100 mg/Kg/day dose group fetuses. Namely, bent limb bones in fetus A010-04 and A024-02 of the control and 100 mg/Kg groups, respectively, and sternoschisis in the above mentioned control fetus (A014-10- that additionally missed a tibia). Due to the single occurrence and group distribution of bent limb bones and sternoschisis, both were considered chance findings.

Among the variations, 7th cervical full ribs were only observed at 1000 mg/Kg bw/day. Three fetuses from three litters had this supernumerary rib at the cervical-thoracic border leading to an incidence of 3.1% per litter. This was above the historical control maximum value (2.7%). Findings related to a 7th cervical full rib are a decreased litter incidence of 14th full ribs, 14th rudimentary ribs, or caudal shift of the pelvic girdle or an increased litter incidence of 7th cervical ossification sites. However, these parameters were considered unaffected by treatment in this study.

The other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within or near the range of available historical control data. Therefore, they were not considered treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 1000 mg/Kg/day.

The only viscerally malformed fetus in this study was a control fetus with ectrodactyly (A014-10). This fetus also had a right-sided aortic arch, diaphragmatic hernia, and its intestines were missing. All these malformations were spontaneous in origin as they accumulated in one control fetus.

Two visceral variations that were observed (small supernumerary liver lobes and convoluted ureter) occurred at low incidences and at frequencies that were within the range of available historical control data. Therefore, they were not considered test treatment-related.
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on fetal anogenital distance (both sexes) observed after treatment up to 1000 mg/Kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose Formulation Analyses

Accuracy

The concentrations analysed in the formulations of Group 2 (100 mg/Kg bw/day), Group 3 (300 mg/Kg bw/day), and Group 4 (1000 mg/Kg bw/day) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test material was observed in the chromatograms of the Group 1 (Control) formulation prepared for use in Week 1. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

 

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 2. Summary of Body Weights (g) – F0 Generation

Post-Coitum

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Day 2

Mean

212

212

209

205

SD

14.5

16.4

15.4

16.5

N

22

22

22

20

 

Day 6

Mean

228

228

225

221

SD

16.9

16.2

16.9

16.7

N

22

22

22

20

 

Day 9

Mean

235

236

232

231

SD

16.5

16.9

17.4

17.5

N

22

22

22

20

 

Day 12

Mean

250

252

248

247

SD

17.0

17.5

17.8

17.8

N

22

22

22

20

 

Day 15

Mean

268

268

265

263

SD

19.0

17.9

19.1

19.2

N

22

22

22

20

 

Day 18

Mean

298

298

295

292

SD

23.1

19.6

22.5

21.5

N

22

22

22

20

 

Day 21

Mean

335

333

331

324

SD

28.3

25.0

28.0

27.8

N

22

22

22

20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3. Summary of Relative Food Consumption (g/Kg Body Weight/Day) – F0 Generation

Post-Coitum

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Days 2-6

Mean

89

85

86

88

SD

7.1

6.3

7.1

7.2

N

22

22

22

20

 

Days 6-9

Mean

81

80

82

85

SD

7.6

7.1

20.1

5.9

N

22

22

22

20

 

Days 9-12

Mean

80

81

82

86*

SD

10.4

7.8

5.9

7.0

N

22

22

22

20

 

Days 12-15

Mean

82

77

81

83

SD

6.1

11.2

7.3

7.2

N

22

22

22

20

 

Days 15-18

Mean

78

76

78

81

SD

6.2

8.0

5.5

7.4

N

22

22

22

20

 

Days 18-21

Mean

70

66

70

70

SD

6.0

5.8

5.5

7.6

N

22

22

22

20

 

Mean of Means

 

80

77

80

82

 

Table 4. Summary of Clinical Biochemistry Parameters Evaluated – F0 Generation

Parameter

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

TSH

uIU/mL

Mean

0.339

0.225

0.241

0.206

SD

0.302

0.141

0.144

0.130

N

22

22

22

20

 

Total T3

ng/dL

Mean

42.0

40.6

38.4

39.1

SD

10.4

10.2

6.8

9.5

N

22

22

22

20

 

Total T4

ug/dL

Mean

2.10

2.06

2.20

1.89

SD

0.58

0.50

0.52

0.54

N

22

22

22

20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. Summary of Macroscopic Findings – F0 Generation

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

END OF TREATMENT

 

 

 

 

Animals examined

22

22

22

22

Animals without findings

21

22

21

21

Animals affected

1

0

1

1

 

Liver

      Right median lobe accessory lobe

1

0

0

0

Thymus

      Focus/foci

0

0

0

1

Spleen

      Alopecia

0

0

1

0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Table 6. Summary of Organ Weights – F0 Generation

Parameter

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

END OF TREATMENT

 

Body Weight (g)

Mean

335

333

331

324

SD

28

25

28

28

N

22

22

22

20

 

Thyroids (g)

Mean

0.0147

0.0151

0.0145

0.0142

SD

0.0024

0.0027

0.0018

0.0022

N

22

22

21

20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 7. Summary of Organ/Body Weight Ratios – F0 Generation

Parameter

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

END OF TREATMENT

 

Body Weight (g)

Mean

335

333

331

324

SD

28

25

28

28

N

22

22

22

20

 

Thyroids (%)

Mean

0.0044

0.0046

0.0044

0.0044

SD

0.0007

0.0008

0.0006

0.0007

N

22

22

21

20

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 8. Summary of Maternal Survival and Pregnancy Status

Dose Group

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

No.

%

No.

%

No.

%

No.

%

Females on study

22

 

22

 

22

 

22

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

0

0.0

0

0.0

0

0.0

0

0.0

    Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

    Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females that were euthanized

0

0.0

0

0.0

0

0.0

0

0.0

    Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

0

0.0

0

0.0

0

0.0

0

0.0

Females examined at scheduled necropsy

22

100.0

22

100.0

22

100.0

22

100.0

    Non gravid

0

0.0

0

0.0

0

0.0

2

9.1

    Gravid

22

100.0

22

100.0

22

100.0

20

90.9

        With Resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

        With Viable fetuses

22

100.0

22

100.0

22

100.0

20

100.0

Totals Females Gravid

22

100.0

22

100.0

22

100.0

20

90.9

 

Table 9. Summary of Fetal Data at Scheduled Necropsy

Group

 

Sex

Viable Fetuses

Dead Fetuses

Resorptions

Post Implantation Loss

Implantation Sites

Corpora Lutea

Pre-Implantation Loss

Fetal Weight in grams

No. of Gravid Females

Male

Female

Early

Late

Group 1

0 mg/Kg/day

Total

113

127

240

0

16

0

16

256

262

6

NA

22

Mean

5.1

5.8

10.9

0.0

0.7

0.0

0.7

11.6

11.9

0.3

5.3

S.D.

1.91

2.79

2.43

0.00

0.88

0.00

0.88

2.36

2.39

0.55

0.27

 

Group 2

100 mg/Kg/day

Total

114

118

232

0

17

1

18

250

268

18

NA

22

Mean

5.2

5.4

10.5

0.0

0.8

0.0

0.8

11.4

12.2

0.8

5.4

S.D.

2.30

1.99

3.05

0.00

1.19

0.21

1.18

2.98

2.34

1.37

0.50

 

Group 3

300

mg/Kg/day

Total

109

124

233

0

9

0

9

242

252

10

NA

22

Mean

5.0

5.6

10.6

0.0

0.4

0.0

0.4

11.0

11.5

0.5

5.3

S.D.

1.33

1.53

1.65

0.00

0.67

0.00

0.67

1.75

1.79

0.51

0.27

 

Group 4

1000

mg/Kg/day

Total

102

105

207

0

9

0

9

216

221

5

NA

20

Mean

5.1

5.3

10.4

0.0

0.5

0.0

0.5

10.8

11.1

0.3

5.3

S.D.

1.92

1.80

1.95

0.00

0.76

0.00

0.76

1.61

1.43

0.55

0.21

None significantly different from control groupNA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA,

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

 

Table 10. Summary of Fetal at Scheduled Necropsy (% per Litter)

Group

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Corpora Lutea

Mean

11.9

12.2

11.5

11.1

S.D.

2.39

2.34

1.79

1.43

N

22

22

22

20

 

Implantation Sites

Mean

11.6

11.4

11.0

10.8

S.D.

2.36

2.98

1.75

1.61

N

22

22

22

20

 

Viable Fetuses (%)

Mean

93.9

92.0

96.5

95.5

S.D.

7.43

11.16

5.59

8.09

N

22

22

22

20

 

Dead Fetuses (%)

Mean

0.0

0.0

0.0

0.0

S.D.

0.00

0.00

0.00

0.00

N

22

22

22

20

 

Early Resorptions (%)

Mean

6.1

7.5

3.5

4.5

S.D.

7.43

11.28

5.59

8.09

N

22

22

22

20

 

Late Resorptions (%)

Mean

0.0

0.5

0.0

0.0

S.D.

0.00

2.13

0.00

0.00

N

22

22

22

20

 

Total Resorptions (%)

Mean

6.1

8.0

3.5

4.5

S.D.

7.43

11.16

5.59

8.09

N

22

22

22

20

 

Pre-implantation Loss (%)

Mean

2.2

7.8

3.9

2.4

S.D.

4.26

14.85

4.58

5.33

N

22

22

22

20

 

Post-implantation Loss (%)

Mean

6.1

8.0

3.5

4.5

S.D.

7.43

11.16

5.59

8.09

N

22

22

22

20

 

Males (%)

Mean

49.8

48.9

46.9

49.7

S.D.

21.61

13.44

11.41

18.05

N

22

22

22

20

 

Females (%)

Mean

50.2

51.1

53.1

50.3

S.D.

21.61

13.44

11.41

18.05

N

22

22

22

20

 

Male Fetal Weights (g)

Mean

5.5

5.5

5.4

5.5

S.D.

0.28

0.53

0.30

0.21

N

22

22

22

20

 

Female Fetal Weights (g)

Mean

5.2

5.2

5.1

5.2

S.D.

0.25

0.45

0.24

0.23

N

21

22

22

19

 

Combined Fetal Weights (g)

Mean

5.3

5.4

5.3

5.3

S.D.

0.27

0.50

0.27

0.21

N

22

22

22

20

 

Male AGD (mm)

Mean

2.77

2.77

2.75

2.82

S.D.

0.284

0.275

0.153

0.235

N

22

22

22

20

 

Female AGD (mm)

Mean

1.29

1.30

1.24

1.33

S.D.

0.315

0.318

0.306

0.300

N

21

22

22

19

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

Table 11. Summary of Fetuses and Litters with Malformations (Absolute Nos)

 

Fetuses

Litters

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Number Examined Externally

240

232

233

207

22

22

22

20

  Ectrodactyly

1

0

0

0

1

0

0

0

  Lower Jaw – Absent or Small

0

0

0

1

0

0

0

1

  Eye – Bulge Absent and/or Small

0

0

0

1

0

0

0

1

 

Number Examined Viscerally

120

116

118

103

22

22

22

20

  Aortic Arch – Right Sided

1

0

0

0

1

0

0

0

  Diaphragmatic Hernia

1

0

0

0

1

0

0

0

  Intestine - Absent

1

0

0

0

1

0

0

0

 

Number Examined Skeletally

121

116

115

104

22

22

22

20

  Bent Limb Bones

1

1

0

1

1

1

0

1

Vertebral Anomaly with or without Rib  Anomaly

1

1

0

1

1

1

0

1

  Orbit(s) – Small

0

1

0

0

0

1

0

0

  Sternochisis

1

0

0

1

1

0

0

1

  Limb bones – Absent

1

0

0

0

1

0

0

0

 

Total Number with Malformations

 

  External

1

0

0

1

1

0

0

1

  Soft Tissue

1

0

0

0

1

0

0

0

  Skeletal

2

2

0

3

2

2

0

3

  Combined

2

2

0

3

2

2

0

3

 

Table 12. Summary of Fetuses and Litters with Variations (Absolute Nos)

 

Fetuses

Litters

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Number Examined Externally

240

232

233

207

22

22

22

20

Number with Findings

0

0

0

0

0

0

0

0

 

Number Examined Viscerally

120

116

118

103

22

22

22

20

  Liver – Small Supernumerary Lobe(s)

1

0

4

2

1

0

4

1

  Ureter(s) – Convoluted

1

1

0

1

1

1

0

1

 

Number Examined Skeletally

121

116

115

104

22

22

22

20

  14thRudimentary Rib(s)

56

66

70

58

19

21

21

17

Reduced Ossification of the Skull

23

7

12

17

12

5

8

10

  Bent Rib(s)

26

22

9

21

12

10

5

12

  Sternebra(e) – Malaligned

17

12

25

14

11

12

16

9

  Pelvic Girdle – Caudal Shift

6

7

9

11

6

6

6

8

Metacarpal(s) and / or Metatarsal(s) Malpositioned

1

0

0

0

1

0

0

0

  14thFull Rib(s)

10

8

10

4

8

6

8

2

Metacarpal(s) and / or Metatarsal(s)  Unossified

3

2

4

2

3

2

4

2

  7thCervical Ossification Site(s)

4

2

3

1

3

2

3

1

Vertebral Centra – Reduced Ossification

2

3

4

0

2

3

3

0

  7thCervical – Full Rib(s)

0

0

0

3

0

0

0

3

Vertebral Arches – Reduced Ossification

0

1

0

1

0

1

0

1

  Scapula(e) - Bent

0

0

0

1

0

0

0

1

  Sternebra(e) #5 and/or #6 Unossified

0

0

0

1

0

0

0

1

Conclusions:
Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rats was determined to be at least 1000 mg/Kg/day.
Executive summary:

A key Guideline OECD 414 developmental toxicity study was conducted to determine the potential of the test material (Neste Renewable Diesel; CAS# 928771-01-1) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats (22/dose). The test material and vehicle (arachis oil) were administered once daily via oral gavage, 7 days a week at doses of 0, 100, 300, 1000 mg/Kg/day from Day 6 to 20 post-coitum, inclusive.

 

Maternal parameters and endpoints evaluated in this study included mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Fetal (F1-generation) parameters determined included the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

 

No treatment-related mortality or changes in any of the maternal parameters investigated (i.e. clinical appearance, body weight, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss) were observed through the study period.

 

At 1000 mg/Kg/day, 3 fetuses of 3 litters were noted with a 7thfull rib. This finding was observed in this high dose group only and the incidence exceeded available historical control data. Therefore, it was considered related to treatment with the test material. However, as this concerns a variation with no known detrimental effects on development, it was considered non-adverse. No other treatment-related changes were observed in any of the developmental parameters investigated in the study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

 

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rats was determined to be at least 1000 mg/Kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-05-18 to 2020-10-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not affect the overall integrity of the study or the interpretation of the study results and conclusions.
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not affect the overall integrity of the study or the interpretation of the study results and conclusions.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviations did not affect the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Neste Renewable Diesel (Neste Oyj (Keilaranta 21, Espoo, PL95, 00095 Neste, Finland); Batch (Lot) Number: K31/NEXBTL-32
- Expiration date of the lot/batch: 2024-06-12
- Purity test date: 2019-06-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: Considered stable for 6 hours at ambient temperature
- Stability under test conditions: Considered stable for 6 hours at ambient temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Stable for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear colourless liquid

OTHER SPECIFICS
- Specific gravity / density: 780.2 kg/m3 at 15°C
- Stability in vehicle: Arachis oil - Stability for at least 6 hours at room temperature under normal laboratory light conditions, for at least 8 days in a refrigerator (2-8°C), and for at least 3 weeks in a freezer (≤ -15°C), has been confirmed over the concentration range 50 to 700 mg/mL (suspensions), project 20223624.
Species:
rabbit
Strain:
New Zealand White
Remarks:
time-mated female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: 3063 and 4348 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet (e.g. ad libitum): On the day of arrival, animals received approximately 25 grams of pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland). During the remainder of the study, the animals received 140-160 grams food per day, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period
- Water (e.g. ad libitum).: Municipal tap water was freely available to each animal via water bottles/containers
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 21°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-05-06; 2020-05-08 To: 2020-10-01
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle (arachis oil) was administered as received. An adequate amount of the vehicle was dispensed into daily aliquots. In the low and mid-dose groups, test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

In the high-dose group, the test material (Neste Renewable Diesel) was administered as a solution as received. An adequate amount of the test material was dispensed into daily aliquots, which were stored the same as for the bulk test material until use.

The test material and test material dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle used in the current study was different than vehicle used in the DRF study (Charles River France Safety Assessment SAS Test Facility Study No. 20210639). This was because the test material formulations prepared with propylene glycol at CRL Den Bosch were considered to be not visually homogeneous. Consequently, an additional trial preparation was performed to select an appropriate vehicle for the main study. These trials were not performed as part of this study and these preparations were not used for dosing. Based on the results, arachis oil was selected for use as a vehicle for preparation of the formulations for the current study.
- Concentration in vehicle: 0, 100, 300, or 780.2 mg/mL for the control, low-dose, mid-dose, and high dose group, respectively
- Amount of vehicle (if gavage): 1 mL/Kg for the control, low-dose, and mid-dose groups and 1.28 mL/Kg for the high dose group, respectively.
- Lot/batch no. (if required): Source: Fagron (Capelle aan de IJssel, The Netherlands)
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method
Analyses were performed by using a validated analytical procedure (Test Facility Study No. 20223624).

- Concentration Analysis
Duplicate sets of middle samples for Groups 1 to 3 (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

- Homogeneity Analysis
Duplicate sets of top, middle and bottom samples for groups 2 and 3 (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

- Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20223624) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20223624.
Details on mating procedure:
Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France). The females arrived on Day
1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
From Day 7 to Day 28 post-coitum
Frequency of treatment:
once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum
Duration of test:
From Day 7 to Day 28 post-coitum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (Arachis Oil - Group 1)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low Dose (Group 2)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid Dose (Group 3)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose (Group 4)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test material.

The dose levels were selected based on the results of a dose range finding study (DRF) performed at Charles River France Safety Assessment SAS (Test Facility Study No. 20210639), in an attempt to produce graded responses to the test material. In this DRF, dose levels of 250, 500 and 1000 mg/Kg/day in propylene glycol were tested. No toxicity was observed up to the highest dose level tested and therefore, dose levels of 100, 300 and 1000 mg/Kg/day were selected for use in the main OECD 414 study. These dose levels were also tested in the OECD 414 study in the rat without apparent toxicity up to 1000 mg/Kg/day.

- Rationale for animal assignment (if not random): At arrival, animals were randomly assigned to groups
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed at least once daily, beginning on Day 7 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. Cage debris was examined to detect abortion or premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations:Animals were individually weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In order to monitor the health status, Animal No. 66 was also weighed on Day 8 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured daily from Day 3 post-coitum onwards.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles / containers

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for animals found dead or sacrificed before planned necropsy)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- The number and distribution of live and dead fetuses
Fetal examinations:
- External examinations: Yes: [all per litter]
For late resorptions and recognizable fetuses in development of females found dead, sacrificed before planned necropsy or that delivered on or before the day of scheduled necropsy, a gross external examination was performed.

Litters of females surviving to scheduled necropsy were subjected to detailed external examinations. Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals found dead or sacrificed before planned necropsy). Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

- Soft tissue examinations: Yes: [all per litter]

Litters of females surviving to scheduled necropsy were subjected to detailed visceral examinations. All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (1984). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar (1972).

The eyes of Fetus A89-03 and livers of Fetus A022-01, A041-02 and A085-08 were collected and fixed in 10% buffered formalin at discretion of the Study Director. The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique (1965). After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

- Skeletal examinations: Yes: [all per litter]
Litters of females surviving to scheduled necropsy were subjected to detailed skeletal examinations.

All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson (1926).

Subsequently, the skeletal examination was done on all fetuses from all groups. All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

- Head examinations: Not specified
Statistics:
For information on statistics, please see section 'Any other information on materials and methods, incl. tables'.
Indices:
Maternal Variables
1) Body Weight Gains: Calculated against the body weight on Day 7 post-coitum.

2) Corrected Body Weight Gains: Body weight determined on Day 29 post-coitum minus the body weight on Day 7 post-coitum and the weight of gravid uterus.

3) Relative Food Consumption: Calculated against the body weight for scheduled intervals

Reproduction and Developmental Variables
1) Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) x 100

2) Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / (number of implantation sites)) x 100

3) Viable fetuses affected/litter (%): (number of viable fetuses affected/litter / number of viable fetuses/litter) x 100
Historical control data:
Historical Control Data is presented in Appendix 3 of the final study report.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed through the study period. Clinical signs that were observed, related to the premature deaths of individual females and have been described in the mortality section below.

Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related mortality was observed during the study period.

In total six animals did not survive until scheduled necropsy as detailed below:

- Female No. 66 (300 mg/Kg dose group) was sacrificed in extremis on Day 8 post-coitum due to absent food consumption for a prolonged period in combination with severe body weight loss. As this mainly occurred prior to start of treatment, this was considered unrelated to treatment.

- Female No. 36 (100 mg/kg/day dose group) was sent to necropsy on Day 25 post-coitum after organic material was found on the manure tray, indicative of a possible early delivery. However, at necropsy, 12 dead fetuses and an early resorption were found in utero. Given the incidental nature and in the absence of a dose relationship, this was considered unrelated to treatment.

The deaths of the following four females were considered related to the gavage procedure and unrelated to treatment.

- Female No. 78 (1000 mg/kg/day dose group) was killed in extremis on Day 16 post coitum. Prior to dosing she was found pale and showed hunched posture. After dosing her posture became abnormal and it was decided to sacrifice this female for animal welfare reasons. At necropsy, foamy contents was observed in the trachea and dark red discoloration and foci were noted in the lungs.

- Female No. 45 (300 mg/kg/day dose group) was killed in extremis on Day 16 post-coitum, shortly after the dosing procedure. Blood was noted on the dosing tube and the female had laboured respiration and rales after dosing. At necropsy, foamy contents was observed in the trachea and a dark red discoloration of the lungs on the left side.

- Female No. 56 (300 mg/kg/day dose group) died shortly after dosing on Day 17 post-coitum. Blood was noted on the dosing tube. At necropsy, foamy contents was observed in the trachea and many dark red foci were noted in the lungs.

- Female No. 2 (Control group) was found dead on Day 18 post-coitum. On Day 17 post-coitum, blood was noted on the dosing tube and laboured respiration was noted. This female remained on study, but was found dead the next day. At necropsy, irregular surface and black/brown discoloration of the right lung was noted. The thoracic cavity contained watery clear fluid. For this female, dosing was omitted on Day 7 post-coitum as blood emerged from the nose on the first day of dosing.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment with the test material. From Day 15 post-coitum onwards, body weight gain was slightly increased at 1000 mg/Kg/day compared to concurrent controls, statistically significant on Day 15 and 21 post-coitum only. Given the direction of change and the minimal size of the effect it was not considered to be treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in food consumption before or after correction for body weight were recorded.

Between Days 12 and 20 post-coitum, food consumption in the control group was slightly reduced compared to previous periods, whereas for the treatment groups food consumption remained similar throughout this part of the treatment period. This resulted in incidental statistically significantly increased food consumption at 300 and 1000 mg/Kg/day. Given the direction of change and the minimal size of the effect it was not considered to be treatment-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable findings. Macroscopic findings related to the premature deaths of individual females have been described in the mortality section above.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre- and post-implantation loss in the control and treatment groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
4 females at 1000 mg/Kg/day were not pregnant at scheduled necropsy compared to 1 in the control and 100 mg/Kg/day groups, and 2 in the 300 mg/Kg/day group. As treatment started after implantation, this was considered a spurious finding and unrelated to treatment with the test material.
Other effects:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites in the control and treatment groups were similar and in the range of normal biological variation.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on fetal body weights (both sexes) were observed up to 1000 mg/Kg/day. Notably, mean fetal weights (male, female and combined) in concurrent controls were slightly lower compared to mean weights in the treatment groups and at the lower end of the range of the historical control data. However, the mean fetal weights were comparable in all treatment groups and were comparable to the historical control mean.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for fetal morphological examination were 179 (20), 179 (20), 144 (17) and 166 (17) in the control, 100, 300 and 1000 mg/Kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/Kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed on litter size of any group.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment with the test material up to 1000 mg/Kg/day.

External malformations occurred in three fetuses from three litters each at the control and high dose level and in one fetus each at 100 and 300 mg/Kg/day.

Malformations observed at the high dose were omphalocele, gastroschisis and distended abdomen (Fetus A074-11, A083-09 and A085-08, respectively). Omphalocele also occurred at 300 mg/Kg/day in one fetus (A052-12) and a distended abdomen was observed for two control fetuses (A019-08 and A020-10). Both omphalocele and distended abdomen have been observed in historical control fetuses Together with the single occurrence of these malformations in the treatment groups, they were considered unrelated to treatment. The fetus with gastroschisis (A083-09) was also malformed viscerally (abnormal liver lobation and opacity of the eyes) and skeletally (vertebral anomaly and sternoschisis) and was as such considered a spurious finding. The malformed fetus at 100 mg/Kg/day (A026-10) had arhinencephaly and a short tail, both with matching skeletal findings. Due to the single occurrence in a low dose fetus, these malformations were considered chance findings.

The remaining control fetus with an external malformation (A012-05) had malrotated hindlimbs without skeletal origin. External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on skeletal morphology were observed following treatment up to 1000 mg/Kg/day.

Besides the anomalies associated with external malformations in fetuses, five different malformations were observed.

The malformations observed at 1000 mg/Kg/day were a costal cartilage anomaly (Fetus A069-07), sternoschisis and vertebral anomaly (both in Fetus A083-09, considered a spurious finding due to multiple anomalies).

A costal cartilage anomaly was also observed in a 100 mg/Kg/day fetus (A030-02) and vertebral anomalies were also noted in a 300 mg/Kg/day (Fetus A047-11) and in five fetuses at 100 mg/Kg/day (A034-01, -11, A038-08, A040-05 and -12).

The two remaining malformations were caudal vertebral anomaly and ectrodactyly with both occurring in 100 mg/Kg fetuses (A026-11 and A028-02, respectively). The high number of vertebral anomalies observed at 100 mg/Kg/day led to a mean litter incidence that was higher than the historical control maximum value (2.2% versus 1.7% per litter, respectively). However, as this was observed in the low dose group, it was considered unrelated to treatment with the test material. Other malformations in this study occurred singly and in the absence of a dose-related response and were therefore considered chance findings.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment with the test material up to 1000 mg/Kg/day. At 1000 mg/Kg/day, besides the multiple malformed fetus A083-09 described earlier, Fetus A079-01 had tetralogy of Fallot. This heart anomaly was also observed in Fetus A052-03 in the 300 mg/Kg/day dose group. Due to the single occurrence and because it is seen previously in historical control fetuses, this finding was considered a chance finding.

The other two malformed fetuses at 300 mg/Kg/day were from Female No. A047. Both fetuses (A047-02 and -06) had abnormal lung lobation and the latter one also had diaphragmatic hernia. The fact that abnormal lung lobulation occurred in only one litter at the mid dose does not indicate a treatment relationship and as both malformations were listed in historical control data, they were considered unrelated to treatment with the test material. The anomalous fetus at 100 mg/Kg/day (A038-06) had malpositioned kidneys that due to the single occurrence was considered a chance finding.

In the control group, one of the fetuses was observed with a distended abdomen (A020-10) and also appeared to have a large atrium. The incidences for absent or small gallbladder at 300 and 1000 mg/Kg/day were significantly increased (statistically) compared to concurrent control. No clear dose response was observed and incidences at 300 and 1000 mg/Kg/day were 2.8 and 2.9% per litter, respectively. The mean values remained within the historical control maximum value (3.1% per litter). Therefore, the increased incidence of absent or small gallbladders was considered unrelated to treatment.

There were increased mean litter incidences of retrocaval ureter in the 300 and 1000 mg/Kg/day groups, but not statistically significant. Retrocaval ureters were observed in 0.5%, 2.6%, 6.5% and 4.9%, per litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. As values were above the historical control upper limit (4.1% per litter) at 300 and 1000 mg/Kg/day, a treatment-related effect could not be excluded. It should be noted, that this variation is the most commonly seen variation in the historical control data. All other variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Dose Formulation Analyses

Accuracy

The concentrations analyzed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. .

 

Homogeneity

The formulations of Group 2 and Group 3 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 2. Summary of Body Weights (F0 Generation)

Sex: Female

 

Post Coitum (Day)

0

7

9

12

15

18

21

24

27

29

Group 1

0 mg/Kg/day

Mean

3743

3670

3710

3780

3864

3897

3905

3952

3990

4022

SD

326.6

302.2

301.7

315.8

303.8

280.4

326.2

327.0

313.4

298.6

N

21

21

21

21

21

20

20

20

20

20

 

Group 2

100 mg/Kg/day

Mean

3618

3579

3619

3681

3785

3828

3840

3872

3933

3988

SD

225.2

201.4

209.9

208.2

224.5

232.0

231.1

236.6

229.3

222.0

N

21

21

21

21

21

21

21

21

20

20

 

Group 3

300

mg/Kg/day

Mean

3595

3548

3578

3650

3753

3830

3834

3894

3952

3989

SD

304.1

256.1

276.3

283.2

287.6

286.7

267.8

254.0

243.9

259.8

N

20

20

19

19

19

17

17

17

17

17

 

Group 4

1000

mg/Kg/day

Mean

3588

3573

3629

3703

3848

3887

3905

3952

3981

4012

SD

305.2

274.1

281.8

388.8

305.2

289.6

274.9

243.9

268.1

266.1

N

18

18

18

18

18

17

17

17

17

17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 3. Summary of Body Weight Gain (%) - F0 Generation

Sex: Female

 

Post Coitum (Day)

7

9

12

15

18

21

24

27

29

Group 1

0 mg/Kg/day

Mean

0

1

3

5

6

6

8

9

10

SD

0.0

0.9

1.5

2.6

3.9

3.7

3.7

4.6

5.4

N

21

21

21

21

21

20

20

20

20

 

Group 2

100 mg/Kg/day

Mean

0

1

3

6

7

7

8

10

12

SD

0.0

1.3

1.7

2.6

2.8

2.9

3.2

3.1

3.1

N

21

21

21

21

21

21

21

20

20

 

Group 3

300

mg/Kg/day

Mean

0

0

2

5

7

7

8

10

11

SD

0.0

2.1

1.9

2.2

2.3

2.6

2.8

3.2

3.5

N

20

19

19

19

19

17

17

17

17

 

Group 4

1000

mg/Kg/day

Mean

0

2

4

8**

8

9*

10

11

12

SD

0.0

0.9

0.9

1.8

2.1

2.3

3.4

4.3

4.9

N

18

18

18

18

18

17

17

17

17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 4. Summary of Relative Food Consumption (g/Kg Body Weight/Day) – F0 Generation

Post-Coitum

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Days 3-4

Mean

39

36

36

40

SD

7.5

12.4

13.6

7.9

N

21

21

20

18

 

Days 4-5

Mean

42

40

40

43

SD

4.3

9.6

10.6

2.8

N

21

21

20

18

 

Days 5-6

Mean

42

42

40

44

SD

6.0

4.1

10.4

2.9

N

21

21

20

18

 

Days 6-7

Mean

41

42

41

44

SD

4.2

3.5

10.1

3.5

N

21

21

20

18

 

Days 7-8

Mean

40

40

37

43

SD

6.7

6.6

11.5

2.8

N

21

21

20

18

 

Days 8-9

Mean

36

38

35

41

SD

9.5

7.3

13.2

4.5

N

21

21

19

18

 

Days 9-10

Mean

37

39

36

42

SD

7.2

5.8

12.0

4.0

N

21

21

19

18

 

Days 10-11

Mean

38

38

36

41

SD

4.6

6.2

8.0

3.8

N

21

21

19

18

 

Days 11-12

Mean

37

36

36

40

SD

6.0

8.2

8.9

6.2

N

21

21

19

18

 

Days 12-13

Mean

33

34

37

39

SD

10.6

10.1

7.2

6.7

N

21

21

19

18

 

Days 13-14

Mean

29

32

29

36

SD

10.9

9.8

11.7

7.2

N

21

21

19

18

 

Days 14-15

Mean

25

32

27

35**

SD

11.5

8.8

12.5

6.0

N

21

21

19

18

 

Days 15-16

Mean

26

32

29

32

SD

12.4

8.8

11.4

11.6

N

21

21

19

18

 

Days 16-17

Mean

28

31

34

35*

SD

11.9

10.6

6.6

7.9

N

20

21

17

17

 

Days 17-18

Mean

29

33

35

35

SD

12.9

7.3

6.8

7.5

N

20

21

17

17

 

Days 18-19

Mean

27

30

35*

35*

SD

12.0

8.3

5.7

7.7

N

20

21

17

17

 

Days 19-20

Mean

28

31

34

34

SD

10.3

6.8

6.3

9.3

N

20

21

17

17

 

Days 20-21

Mean

31

31

32

33

SD

6.9

7.7

7.4

7.8

N

20

21

17

17

 

Days 21-22

Mean

32

30

32

30

SD

7.2

6.5

7.3

7.6

N

20

21

17

17

 

Days 22-23

Mean

28

30

30

29

SD

7.8

6.7

6.7

7.2

N

20

21

17

17

 

Days 23-24

Mean

26

29

28

27

SD

7.5

5.2

7.1

8.1

N

20

21

17

17

 

Days 24-25

Mean

24

27

27

25

SD

8.6

8.8

6.7

7.8

N

20

21

17

17

 

Days 25-26

Mean

24

28

25

25

SD

8.0

8.5

10.4

8.4

N

20

20

17

17

 

Days 26-27

Mean

23

28

27

25

SD

7.7

7.2

9.0

7.7

N

20

20

17

17

 

Days 27-28

Mean

22

28*

28

25

SD

8.8

8.5

6.9

9.6

N

20

20

17

17

 

Days 28-29

Mean

23

28

27

27

SD

8.5

6.3

10.0

9.0

N

20

20

17

17

Mean of Means

 

31

33

33

35

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 5. Summary of Maternal Survival and Pregnancy Status

Dose Group

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

No.

%

No.

%

No.

%

No.

%

Females on study

22

 

22

 

22

 

22

 

Females that aborted or delivered

0

0.0

0

0.0

0

0.0

0

0.0

Females that died

1

4.5

0

0.0

1

4.5

0

0.0

    Females that aborted

0

0.0

0

0.0

0

0.0

0

0.0

    Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

1

100.0

0

0.0

1

100.0

0

0.0

Females that were euthanized

0

0.0

1

4.5

2

9.1

1

4.5

    Non gravid

0

0.0

0

0.0

0

0.0

0

0.0

    Gravid

0

0.0

1

100.0

2

100.0

1

100.0

Females examined at scheduled necropsy

21

95.5

21

95.5

19

86.4

21

95.5

    Non gravid

1

4.8

1

4.8

2

10.5

4

19.0

    Gravid

20

95.2

20

95.2

17

89.5

17

81.0

        With Resorptions only

0

0.0

0

0.0

0

0.0

0

0.0

        With Viable fetuses

20

100.0

20

100.0

17

100.0

17

100.0

Totals Females Gravid

21

95.5

21

95.5

20

90.9

18

81.8

 

Table 6. Summary of Fetal Data at Scheduled Necropsy

Group

 

Sex

Viable Fetuses

Dead Fetuses

Resorptions

Post Implantation Loss

Implantation Sites

Corpora Lutea

Pre-Implantation Loss

Fetal Weight in grams

No. of Gravid Females

Male

Female

Early

Late

Group 1

0 mg/Kg/day

Total

93

86

179

1

6

13

20

199

227

28

NA

20

Mean

4.7

4.3

9.0

0.1

0.3

0.7

1.0

10.0

11.4

1.4

37.5

S.D.

2.35

2.13

2.21

0.22

0.73

1.27

1.62

2.58

1.79

2.19

5.27

 

Group 2

100 mg/Kg/day

Total

84

95

179

1

10

10

21

200

237

37

NA

20

Mean

4.2

4.8

9.0

0.1

0.5

0.5

1.1

10.0

11.9

1.9

38.8

S.D.

1.54

1.62

2.37

0.22

0.95

0.95

1.61

2.00

2.32

1.87

4.09

 

Group 3

300

mg/Kg/day

Total

71

73

144

0

6

28

34

178

191

13

NA

17

Mean

4.2

4.3

8.5

0.0

0.4

1.6

2.0

10.5

11.2

0.8

38.6

S.D.

1.70

1.90

2.62

0.00

0.61

2.34

2.50

1.62

1.25

0.97

4.79

 

Group 4

1000

mg/Kg/day

Total

76

90

166

0

8

6

14

180

191

11

NA

17

Mean

4.5

5.3

9.8

0.0

0.5

0.4

0.8

10.6

11.2

0.6

38.8

S.D.

1.66

1.45

1.44

0.00

0.62

0.70

1.24

1.46

1.68

0.86

4.00

None significantly different from control groupNA = NOT APPLICABLE

MEAN NUMBER OF VIABLE FETUSES, MEAN NUMBER OF IMPLANTATION SITES, MEAN NUMBER OF CORPORA LUTEA, FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

 

Table 7. Summary of Fetal at Scheduled Necropsy (% per Litter)

Group

 

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Corpora Lutea

Mean

11.4

11.9

11.2

11.2

S.D.

1.79

2.32

1.25

1.68

N

20

20

17

17

 

Implantation Sites

Mean

10.0

10.0

10.5

10.6

S.D.

2.58

2.00

1.62

1.46

N

20

20

17

17

 

Viable Fetuses (%)

Mean

91.3

89.4

81.2

92.7

S.D.

13.26

15.87

23.73

10.18

N

20

20

17

17

 

Dead Fetuses (%)

Mean

0.5

0.3

0.0

0.0

S.D.

2.03

1.50

0.00

0.00

N

20

20

17

17

 

Early Resorptions (%)

Mean

2.5

4.8

3.6

4.4

S.D.

5.64

8.84

6.41

5.65

N

20

20

17

17

 

Late Resorptions (%)

Mean

5.7

5.5

15.2

2.9

S.D.

10.99

11.22

22.64

5.65

N

20

20

17

17

 

Total Resorptions (%)

Mean

8.2

10.3

18.8

7.3

S.D.

12.73

15.74

23.73

10.18

N

20

20

17

17

 

Pre-implantation Loss (%)

Mean

11.9

14.8

6.9

5.4

S.D.

19.16

13.08

8.68

6.63

N

20

20

17

17

 

Post-implantation Loss (%)

Mean

8.7

10.6

18.8

7.3

S.D.

13.26

15.87

23.73

10.18

N

20

20

17

17

 

Males (%)

Mean

52.0

46.9

50.1

45.4

S.D.

21.08

13.28

16.55

14.04

N

20

20

17

17

 

Females (%)

Mean

48.0

53.1

49.9

54.6

S.D.

21.08

13.28

16.55

14.04

N

20

20

17

17

 

Male Fetal Weights (g)

Mean

38.5

39.6

38.7

39.9

S.D.

4.53

5.42

5.72

5.69

N

20

20

17

17

 

Female Fetal Weights (g)

Mean

37.5

38.7

38.5

38.1

S.D.

6.24

4.01

4.80

3.79

N

20

20

16

17

 

Combined Fetal Weights (g)

Mean

37.5

38.8

38.6

38.8

S.D.

5.27

4.09

4.79

4.00

N

20

20

17

17

PROPORTIONAL (%) DATA COMPARED USING THE MANN-WHITNEY TEST

FETAL WEIGHTS COMPARED USING DUNNETT'S TEST

None significantly different from control group

 

Table 8. Overview of Litter Incidence (absolute numbers and % per litter) of External Malformations Observed

Findings

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Historical Control Data (HCD)

Omphalocele

-

-

1 fetus; 1 litter

0.5 % per litter

1 fetus; 1 litter

0.5 % per litter

Mean = 0.2

Max = 3.0

% per litter

 

Gastroschisis

-

-

-

1 fetus; 1 litter

0.7% per litter

Not Present

 

Distended abdomen

2 fetuses;

2 litters

0.9% per litter

-

-

1 fetus; 1 litter

0.7% per litter

Mean = 0.0

Max = 0.6

% per litter

 

Arhinencephaly

-

1 fetus; 1 litter

0.5 % per litter

-

-

Not Present

 

Short tail

-

1 fetus; 1 litter

0.5 % per litter

-

-

Mean = 0.0

Max = 0.4

% per litter

- = not observed in this group

 

Table 9. Overview of Litter Incidence (absolute numbers and % per litter) of Visceral Malformations Observed

Findings

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Historical Control Data (HCD)

Abnormal lobulation

lung

-

-

2 fetuses; 1 litter

1.3% per litter

-

Mean = 0.1

Max = 2.7

% per litter

 

Diaphragmatic

hernia

-

-

1 fetus; 1 litter

0.7 % per litter

-

Mean = 0.0

Max = 0.6

% per litter

 

Tetralogy of fallot

-

-

1 fetus; 1 litter

0.5% per litter

1 fetus; 1 litter

0.7% per litter

Mean = 0.1

Max = 0.8

% per litter

 

Malpositioned

kidneys

-

1 fetus; 1 litter

0.5 % per litter

-

-

Mean = 0.1

Max = 0.9

% per litter

 

Abnormal lobulation

liver

-

-

-

1 fetus; 1 litter

0.7 % per litter

Mean = 0.1

Max = 0.6

% per litter

 

Eye opacity

-

-

-

1 fetus; 1 litter

0.7 % per litter

Not Present

- = not observed in this group

 

Table 10. Overview of Litter Incidence (absolute numbers and % per litter) of Skeletal Malformations Observed

Findings

Group 1

0 mg/Kg/day

Group 2

100 mg/Kg/day

Group 3

300

mg/Kg/day

Group 4

1000

mg/Kg/day

Historical Control Data (HCD)

Vertebral anomaly

-

5 fetuses; 3

litters

2.2% per litter

1 fetus; 1 litter

0.7 % per litter

1 fetus; 1 litter

0.7 % per litter

Mean = 0.5

Max = 1.7

% per litter

 

Costal cartilage

anomaly

-

1 fetus; 1 litter

0.7 % per litter

-

1 fetus; 1 litter

0.7 % per litter

Mean = 0.0

Max = 0.6

% per litter

 

Caudal vertebral

anomaly

-

1 fetus; 1 litter

0.5 % per litter

-

-

Mean = 0.1

Max = 0.7

% per litter

 

Ectrodactyly

-

1 fetus; 1 litter

0.4 % per litter

-

-

Not Present

 

Sternoschisis

-

-

-

1 fetus; 1 litter

0.7 % per litter

Not Present

- = not observed in this group


Conclusions:
Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rabbits was determined to be at least 1000 mg/Kg/day.
Executive summary:

A key Guideline OECD 414 developmental toxicity study was conducted to determine the potential of the test material (Neste Renewable Diesel; CAS# 928771-01-1) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits. The test material and vehicle (arachis oil) were administered once daily via oral gavage, 7 days a week at doses of 0, 100, 300, 1000 mg/Kg/day from Day 7 to Day 28 post-coitum, inclusive.

 

Maternal parameters and endpoints evaluated in this study included mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. Fetal (F1-generation) parameters determined included the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral, and skeletal malformations and developmental variations.

 

No treatment-related mortality or changes in any of the maternal parameters investigated (i.e. clinical appearance, body weight, food consumption and macroscopic examination) were observed through the study period.

 

Six females did not survive until scheduled necropsy (one in the control group, one at 100 mg/Kg/day, three at 300 mg/Kg/day, and one at 1000 mg/Kg/day). As these premature deaths were considered to have occurred either as a result of the gavage procedure, symptoms occurring prior to start of the treatment, or were considered incidental, they were determined not to be treatment-related.

 

Increased mean litter incidences of retrocaval ureter were observed in the 300 and 1000 mg/Kg/day dose groups. The incidences for retrocaval ureter were not significantly increased (statistically) and had no apparent dose-response. However, as values were above the historical control maximum value at 300 and 1000 mg/Kg/day, a treatment-related effect could not be excluded. As this concerns a variation with no detrimental effects on development, it was considered to be non-adverse.

 

No treatment-related changes were observed in any of the remaining developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral, and skeletal malformations and developmental variations).

 

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rabbits was determined to be at least 1000 mg/Kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
other: Rat and Rabbit
Quality of whole database:
Two Guideline OECD 414 studies, one in rodents and one in non rodents available for assessment.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A key Guideline OECD 414 developmental toxicity study (Charles River Laboratories Den Bosch BV, 2020) was conducted to determine the potential ofthe test material (Neste Renewable Diesel; CAS# 928771-01-1) to induce developmental toxicity aftermaternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats (22/dose). The test material and vehicle (arachis oil) were administered once daily via oral gavage, 7 days a week at doses of 0, 100, 300, 1000 mg/Kg/day from Day 6 to 20 post-coitum, inclusive.

 

Maternal parameters and endpoints evaluated in this study included mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. Fetal (F1-generation) parameters determined included the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

 

No treatment-related mortality or changes in any of the maternal parameters investigated (i.e. clinical appearance, body weight, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss) were observed through the study period.

 

At 1000 mg/Kg/day, 3 fetuses of 3 litters were noted with a 7thfull rib. This finding was observed in this high dose group only and the incidence exceeded available historical control data. Therefore, it was considered related to treatment with the test material. However, as this concerns a variation with no known detrimental effects on development, it was considered non-adverse. No other treatment-related changes were observed in any of the developmental parameters investigated in the study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

 

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for NesteRenewable Diesel in rats was determined to be at least 1000 mg/Kg/day.

A second key Guideline OECD 414 developmental toxicity study ( Charles River Laboratories Den Bosch BV, 2020) was conducted to determine the potential of the test material (Neste Renewable Diesel; CAS# 928771-01-1) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits. The test material and vehicle (arachis oil) were administered once daily via oral gavage, 7 days a week at doses of 0, 100, 300, 1000 mg/Kg/day from Day 7 to Day 28 post-coitum, inclusive.

 

Maternal parameters and endpoints evaluated in this study included mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. Fetal (F1-generation) parameters determined included the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral, and skeletal malformations and developmental variations.

 

No treatment-related mortality or changes in any of the maternal parameters investigated (i.e. clinical appearance, body weight, food consumption and macroscopic examination) were observed through the study period.

 

Six females did not survive until scheduled necropsy (one in the control group, one at 100 mg/Kg/day, three at 300 mg/Kg/day, and one at 1000 mg/Kg/day). As these premature deaths were considered to have occurred either as a result of the gavage procedure, symptoms occurring prior to start of the treatment, or were considered incidental, they were determined not to be treatment-related.

 

Increased mean litter incidences of retrocaval ureter were observed in the 300 and 1000 mg/Kg/day dose groups. The incidences for retrocaval ureter were not significantly increased (statistically) and had no apparent dose-response. However, as values were above the historical control maximum value at 300 and 1000 mg/Kg/day, a treatment-related effect could not be excluded. As this concerns a variation with no detrimental effects on development, it was considered to be non-adverse.

 

No treatment-related changes were observed in any of the remaining developmental parametersinvestigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral, and skeletal malformations and developmental variations).

 

Based on the lack of adverse treatment-related effects observed in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Neste Renewable Diesel in rabbits was determined to be at least 1000 mg/Kg/day.

Additionally, no statistically significant or treatment related effects were found ion number of corpora lutea, implantation sites or number of live births in pregnant female rats administered NExBTL renewable diesel over 2-generations at dose levels of 50, 250 or 1000 mg/Kg bw/day (Harlan Laboratories Ltd., 2009). The study, which was GLP-compliant and included a sub-chronic reproductive toxicity phase, followed OECD guideline 416. An approx. 8-10% reduction in mean litter size for the intermediate and high dose groups was considered a chance finding of negligible toxicological significance an overall oral NOAEL of 1000 mg/Kg bw/d for developmental toxicity. The study was conducted to achieve compliance under chemical, safety and health regulations other than EU REACH.

Justification for classification or non-classification

Based on the results of the available studies, the substance does not meet the criteria for classification as a reproductive or developmental toxicant under Regulation EC 1272/2008.

Additional information