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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/D evelopmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28- day oral toxicity study in rodents
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,7,11,15-tetramethylhexadecane-1,2,3-triol
EC Number:
277-923-2
EC Name:
3,7,11,15-tetramethylhexadecane-1,2,3-triol
Cas Number:
74563-64-7
Molecular formula:
C20H42O3
IUPAC Name:
3,7,11,15-tetramethylhexadecane-1,2,3-triol
Test material form:
liquid: viscous
Details on test material:
- Physical appearance: colourless viscous liquid
- Storage conditions: at room temperature

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Details on species / strain selection:
Untreated, nulliparous, non-pregnant females and untreated males were used at the initiation of the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Age at start pretest: Females: approximately 10-12 weeks; Age at start F0-tre
atment: Males approximately 10-12 weeks, Females approximately 12-14 weeks
- Weight at study initiation: males 249-280 g; females 191-231 g
- Fasting period before study: no
- Housing: pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV
type, height 18 cm); Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon
plastic cages (MIV type, height 18 cm); Mating: Males and females were cohabitated on a 1:1 basis
in Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Males were housed in their home
cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females
were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Females wer
e housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except
during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage
using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without
their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Spezialdiäten GmbH, Soest, Germany) (During motor activity measurements, animals did not have
access to food for a maximum of 1.5 hours)
- Water: Free access to tap-water (During motor activity measurements, animals did not have access
to water for a maximum of 1.5 hours)
- Acclimation period: Females: At least 5 days prior to start of treatment;
Males: Two days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting
material evaluation for contaminants and/or nutrients was performed according to facility standard
procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 October 2016 To: 23 December 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/ composition of the test item.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum.
- A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of samples of formulations taken on a single occasion during the treatment phase were analysed according to a validated method. The samples were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study.
Duration of treatment / exposure:
Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 41-54 days. Routinely, females that are littering are left undisturbed.The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily for 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Based on the results of a 14-day dose range finding study the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg bw/day. In this DRF increased liver weights only was observed at 500 mg/kg bw/day after 14 days of treatment and mortality occurred within 9 days after treatment at 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:
Observations and examinations performed and frequency

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, 19 and 20 postcoitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION:
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: F0 females 6h after dosing on Day 14 of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, o/n (with a maximum of 24h)
- How many animals: 5 F0/sex/group
- Parameters checked according to Guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: F0 females 6h after dosing on Day 14 of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, o/n (with a maximum of 24h)
- How many animals: 5 F0/sex/group
- Parameters checked according to Guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillar reflex and static righting reflex, fore- and hind-limb grip strength, locomotor activity.

IMMUNOLOGY: No

OTHER: reproduction and developmental parameters, plasma thyroid hormone analysis
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those
females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, staging of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma thyroid hormone level.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/ determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the mating period (a minimum of 28 days of dose administration).
- Females which deleivered: PND 14-16.
- Females which failed to deliver: Post-coitum Days 25-26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
- Females with total litter loss: Within 24 hours of litter loss.

GROSS NECROPSY
- Gross necropsy according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
According to Guidelines
Postmortem examinations (offspring):
GROSS NECROPSY
-All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered
formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%) = (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%) = (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pale faeces were temporary observed in two females at 500 mg/kg bw/day during the post coitum phase. A relation to treatment of this finding could not be ruled out. However, based on the incidence (in 2/10 females) and absence of other signs of ill health in these females, this finding was considered non-adverse and of no toxicological significance. Salivation seen in a high incidence after dosing among animals of the 150 and 500 mg/kg bw/day dose groups for prolonged periods during treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. immediately after dosing). Salivation was also observed occasionally in animals of the control and the 50 mg/kg bw/day dose group. Incidental findings that were noted included rales, piloerection, scabs (on various locations in the animals) and alopecia. These findings occurred within the range of background findings to be expe cted for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male at 150 mg/kg bw/day died after blood sampling shortly before scheduled sacrifice. Its death was considered to be accidental and not related to treatment.
No further mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant lower body weights and body weight gain were observed in males treated at 500 mg/kg bw/day on all occasions after start of treatment until sacrifice four weeks later. A 22% body weight gain was observed in these males of the treatment period, compared to 34% body weight gain in the controls. Body weights and body weight gain in the 50 and 150 mg/kg bw/day treated males remained in the same range as controls over the whole treatment period.
In females treated at 500 mg/kg bw/day, body weights and body weight gain were also lower until start of mating, two weeks after start of treatment, achieving levels of statistical significance for body weight gain on day 1 of mating in comparison with controls. During pregnancy any treatment related effect on maternal body weight and body weight gain was obscured by the increase in body weight due to their pregnancy. Nevertheless, lower body weights and body weight gain were observed during the post coitum phase in high dose females, achieving levels of statistical significance for body weights on day 17 and 20 post coitum, in comparison with controls. During lactation, statistically lower body weights were observed in the high dose dams from Day 1 until Day 13, in comparison with controls. Body weight gain, however, showed that their growth was normal and ran parallel to that of the other groups in the lactation phase.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption was observed in males at 500 mg/kg bw/day over the first week of treatment (days 1-8 premating). During the continuation of treatment until sacrifice four week later, the food consumption before and after allowance for body weight of these high dose males was within the same range as of the other groups.
In females at 500 mg/kg bw/day, food consumption was (minimally) lower on all occasions from start of treatment until sacrifice at the end of lactation, achieving levels of statistical significance for absolute food consumption over days 0-4, 14-17 and 17-20 post coitum and days 4-7 and days 7-13 of lactation when compared to controls. Statistical significance was also reached for the relative food consumption in high dose females over days 7-13 of lactation.
Food consumption before or after allowance for body weight in males and females treated at 50 and 150 mg/kg bw/day was considered normal and in the similar range as the respective controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haematological parameters of treated male and female rats were considered not to be affected after treatment up to 500 mg/kg bw/day.
The statistical significance for the mean red blood cell count in males at 500 mg/kg bw/day was considered to have occurred as a result of the relatively low control mean red blood cell count in comparison to the historical background data.
Any statistically significant changes for monocytes in low dose males and red blood cells and haematocrit in mid dose females were considered not to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Increases in the mean enzyme levels ALAT and ASAT were observed in male and female rats at 500 mg/kg bw/day, achieving levels of statistical significance for ALAT in both sexes and for ASAT in males when compared to controls.
- A statistically significant decrease in the mean level for total protein in males at 500 mg/kg bw/day.
The total protein levels in females at 500 mg/kg bw/day were considered not to be affected by treatment. The lower mean value observed in these females was the result of a low protein level of 37.4 g/ L (and concurrent low albumin level) in a single female which was considered to be a fortuitous finding and not representative of an effect in the group.
- A dose related increase in mean glucose level was observed in male rats at 150 and 500 mg/kg bw/day, not reaching statistical significance. The increase was minimal and considered non-adverse, since the glucose levels, even in the males at 500 mg/kg bw/day, were within (the high end of) the normal range for male rats of this age and strain.
The statistical significance for mean glucose levels in females at 150 and 500 mg/kg bw/day were the result of a relative low mean glucose level in controls. Since all glucose values remained within the range considered normal for female rats of this age and strain and in the absence of a treatment related distribution, this finding was considered not to be toxicologically relevant.
No further toxicologically relevant changes occurred in clinical biochemistry parameters of treated male and female rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.
A tendency to a dose related increase in fore limb grip strength was observed in both sexes, not reaching levels of statistical significance. Since the grip strength values even in the high dose group at 500 mg/kg bw/day remained within normal limits for rats of this strain and age, this finding was considered to be non-adverse. The hind limb grip strength in the test item treated groups remained in the same range as controls.
For the motor activity, slightly higher mean values for total movements and ambulations were observed in high dose males at 500 mg/kg bw/day. These increases were the result of different habituation profile in these males, expressed by a higher activity of these males during the first part of the measurement (i.e. during the first five 5-minute intervals), in comparison with the habituation of the males in the other groups. The magnitude of change in habituation profile was minimal since the values of total movements and ambulations remained within normal limits for rats of this strain and age. Therefore this finding was considered to be nonadverse.
The variation in motor activity in test item treated females did not indicate a relation with treatment and remained in the same range as controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Phytantriol were noted in the stomach of the 500 mg/kg bw/day group males (minimal/slight hyperplasia of the squamous cells, and minimal/slight edema) and the liver of the 500 mg/kg/bw day females (minimal/slight hepatocellular hypertrophy).
In the kidneys, a minimally increased severity of hyaline droplet accumulation was present in all treated males compared to control animals. In the absence of a dose response and since these severities are within the normal background range, this was considered incidental and not test item-related.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
During the pretest and premating period, all females had regular cycles of 4 days, except for a single cycle of 5 days in one control female. During mating, no mating was confirmed in three females (one at 50 mg/kg bw/day, one at 150 mg/kg bw/day and one at 500 mg/kg bw/day) and (extended) di-estrus was recorded in their smears. Reanalysis of the smears that were prepared during the mating period of these three females showed presence of sperm on day 2 of mating in female of dose group 150 mg/kg bw/day, which was in agreement with the fact that delivery was recorded for this female approximately three weeks after start of mating, and on day 5 of mating in female dosed at 50 mg/kg bw/day. This low dose female was therefore considered to have mated. No evidence of mating was obtained from the smears in the female from the high dose group.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating index: Mating index was considered not to be affected by treatment. One female at 500 mg/kg bw/day showed no evidence of mating. In two other females (one at 50 mg/kg bw/day and one at 150 mg/kg bw/day) mating was overlooked during the mating period, but re-analysis of the smears at a later stage revealed sperm in one of the smears of each of the females indicating that mating had occurred. Whereas delivery of a litter was recorded for one female dosed 150 mg/kg bw/day, thus confirming its mating, mating of female dosed 50 mg/kg bw/day but did not result in a pregnancy.

Fertility index: Fertility index was considered not to be affected by treatment. In four females (two at 50 mg/kg bw/day and two at 150 mg/kg bw/day) mating did not result in pregnancy. In the absence of a dose-response relationship in the fertility index, the incidences of nonpregnancy after mating were considered to have occurred accidentally and no toxicological significance was attached to this finding. Moreover, the number of non pregnant females after mating in each group was within the historical background data range (Historical background range for the fertility index: mean 94, st.dev. 8.1, min 80-max 100, N = 35).

Precoital time: Precoital time was considered not to be affected by treatment.

Number of implantation sites: A dose related decrease in the number of implantation sites was apparent, being statistically significantly lower than controls in females at 500 mg/kg/day. The mean number of implantations sites were 13.9, 12.4, 10.5 and 9.6 for the controls, 50, 150 and 500 mg/kg bw/day dose group, respectively. It should be noted that the mean value of the 150 mg/kg bw/day dose groups was largely affected by two females with very low number of implantation sites, i.e.one female with with 1 implantation site only (no offspring) and one female with 3 implantation sites (the single pup was lean at birth and went missing on lactation day 3). In the 500 mg/kg bw/day dose group, there was one female with a very low number of implantation sites (no offspring) but also more general reduction of the mean number of implantation sites in the other females in this group. A relatively high mean number of implantation sites was observed in the controls compared to historical background data for rats of this strain and age (mean 12.3, st.dev 0.92, min 10.7-max 14.2, N = 26), Nevertheless, the mean number of implantation sites in the females of the 150 and 500 mg/kg bw/day treated groups were outside the historical range.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced number of implantations with a corresponding reduced mean number of living pups per litter

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
For two out of four pups that went missing shortly after birth it was recorded that they were lean, which was also confirmed by their relatively low body weight recorded on PND 1. Hypothermia and the absence of milk in the stomach was additionally observed in one of these two pups.
Incidental clinical symptoms in pups consisted of small size, missing tail apex, blue spots on tail, blue and white staining of tail apex and pale appearance. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mean number of living pups per litter at first litter check was statistically significantly lower in the 500 mg/kg bw/day group, when compared to controls. The mean number of live pups/litter on PND 1 was 13.3, 11.5, 11.4 and 9.8 for the vehicle, 50, 150 and 500 mg/kg bw/day Group, respectively. This reduction of number of pups per litter in the high dose group was the direct result of the lower number of implantation sites observed at this dose level. Similar to that observed for the mean number of implantations sites, a relatively high mean number of living pups at first litter check was observed in the controls compared to historical background data for rats of this strain and age (mean 11.4, st.dev 1.18, min 8.8-max 13.3, N = 26). The mean number of living pups in the treated groups remained within the range of historical background data for rats of this strain and age.
However, the post-implantation index, i.e. the total number of offspring born compared to the total number of uterine implantations, was considered not to be affected by treatment. The post-implantation survival index was 96, 93, 95 and 91% for the vehicle, 50, 150 and 500 mg/kg bw/day Group, respectively.
For one control female, one mid dose female (150 mg/kg bw/day) and one high dose female (500 mg/kg bw/day), the number of pups were slightly higher than the number of implantations. This phenomenon is observed from
time to time and is caused by normal resorption of these areas during (the 14-16 days of) lactation. No toxicological relevance was attached to this finding in the current study.

The number of live offspring at first litter check PND 1 compared to the total number of offspring born was considered not to be affected by treatment. The live birth index was 99, 100, 100 and 100% for the vehicle, 50, 150 and 500 mg/kg bw/day Group, respectively. One pup of a control dam was found dead at first litter check. No toxicological relevance was attributed to this dead pup since it had occurred in a single case and remained within the range considered normal in litters of rats of this strain.

The number of live offspring before culling on PND 4 compared to the number of offspring at first litter check on PND 1 was considered not affected by treatment.
Four pups (one from a control litter, two from separate litters at 50 mg/kg bw/day and one from a litter at 150 mg/kg bw/day) went missing on PND 2 or 3. These pups were most likely cannibalised. The missing pup at 150 mg/kg bw/day was the only offspring in that litter and thus meant total litter loss for its dam. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of pups in litters of the test groups at first litter check on PND 1 were in the same range as controls. During lactation, the growth of the pups from litters at 500 mg/kg bw/day was retarded, which resulted in statistically significantly lower body weights for both the male and female pups on PND 13, approximately 11% lower than in controls. The body weight of pups in litters at 50 and 150 mg/kg bw/day remained in the same range as controls over the whole lactation period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
The statistical significances that were apparent for the mean T4 levels for the female pups in all three test groups were considered to be the results of an accidentally high mean control T4 level in comparison with the historical background data. Therefore, no toxicological significance was attached to this finding.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. The statistical significances apparent for the corrected anogenital distance of male pups at 50
and 150 mg/kg bw/day occurred without a dose related-trend and was attributed to a relatively high intra-group differences. These variations were considered not to be related to treatment.
Treatment up to and including 500 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
other: growth retardation during lactation
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Accuracy:

In the control group, no test item was detected.

The concentrations analysed in the formulations of low, md and high dose groups were between 96% and 98% (in agreement with target concentrations, i.e. mean accuracies between 90% and 110%).

Homogeneity:

The formulations in the low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 10%), with coefficients of variation of 1.2% and 0.26% in the low and high dose, respectively.

Applicant's summary and conclusion

Conclusions:
Based on reduced body weight gain and reduced food consumption observed at 500 mg/kg bw/day, a NOAEL of 150 mg/kg bw/day is derived for parental toxicity.
Treatment-related effects in reproduction were observed as reduced number of implantation sites at 150 and 500 mg/kg bw/day, being statistically significant only at 500 mg/kg bw/day. A corresponding reduction of mean number of living pups per litter was observed at 500 mg/kg bw/day only. Retardation of growth of pups during lactation was observed at 500 mg/kg bw/day. Based on these results a NOAEL reproduction toxicity and a NOAEL developmental toxicity of 150 mg/kg bw/day was established.
Executive summary:

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPFbred Wistar Han rats in a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test. One control group and three treated groups were tested (50, 150, 500 mg/kg bw/day), each consisting of 10 males and 10 females. Males were treated for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 41-54 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility, precoital time,

number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy). Blood was sampled for exposure control evaluation (from first 5 F0-males and all F0-females/sex/group at day 14 of treatment and from PND 13-15 pups from litters of the selected 5 F0-females). Accuracy and homogeneity of formulations were demonstrated by analyses.

Parental results:

After treatment at 500 mg/kg bw/day, a minimal increase in fore limb grip strength was observed in both sexes and a slightly different habituation profile in males only, as expressed by a higher number of movements and ambulations during the first part of motor activity testing. Due to the low magnitude of the changes in grip strength and motor activity, the results were still within the normal limits for these parameters in rats of this age and strain. Therefore, these findings at the dose level of 500 mg/kg bw/day were considered to be non-adverse.

Treatment-related changes at 500 mg/kg bw/day were observed as statistically significant lower body weights and food consumption, changes in several clinical biochemistry parameters, including increases in ALAT and ASAT, and glucose and a decrease in total protein. The changes in glucose and total protein were considered to be non-adverse, based on the low magnitude of change. Minimal increases in liver weights were observed in these high dose animals, still remaining within normal limits. In addition, a low severity of hepatocellular hypertrophy was recorded in the females at 500 mg/kg bw/day, which was considered to be a non-adverse finding in the absence of any degenerative findings in the liver of these animals. Whereas the increases in the enzyme levels ALAT and ASAT and liver weights were slightly more pronounced in males than in females, the hepatocellular hypertrophy was only seen in females. Although the increased enzyme levels might be indicative of liver damage, this was not confirmed by histopathology in the males. Therefore, also the changes in enzyme levels and liver weight were considered to be non-adverse.

Histopathological examination revealed squamous cell hyperplasia and edema in the stomach of males at 500 mg/kg bw/day, which were considered to represent non-adverse mild local reaction to the test item. No treatment-related changes were noted after treatment at 500 mg/kg bw/day in the other parental parameters investigated in this study (i.e. clinical appearance, haematology investigations, sperm staging (in males) and estrous cycle (in females)). Any changes in the various study parameters after treatment at 50 and 150 mg/kg bw/day were considered no signs of treatment related parental toxicity.

Reproductive results:

Incidental occurrences of non-mated and non-pregnant females and females with implantation sites only and/or a female with total litter loss were noted among the test groups treated with Phytantriol, but none in the vehicle control group. The incidence of each finding was within normal limits for rats of this strain and age and the distribution of each of these findings over the test groups did not indicate an immediate relation to treatment. Moreover, the overall distribution of the females with litters comprising a normal number of pups was 10, 8, 6 and 8 for the control, 50, 150 and 500 mg/kg bw/day treated groups respectively. These results also did not indicate a dose effect relationship. A reduction was observed in the mean number of implantation sites at 150 and 500 mg/kg bw/day reaching statistical significance only at the high dose. Whereas the mean number of implantation sites were below the lower limit of the historical background data in females treated at 150 mg/kg bw/day (minimally lower) and 500 mg/kg bw/day, the mean number of living pups per litter was not affected at 150 mg/kg bw/day and only slightly decreased at 500 mg/kg bw/day. The mean number of living pups per litter remained within the historical range in all dose groups. No corroborating effects (of toxicity) were observed in either the parental females or the offspring in the 150 mg/kg bw/day dose group.

Developmental results:

After treatment at 500 mg/kg bw/day, a lower number of pups per litter were observed, which was directly correlated to the lower number of implantation sites in the corresponding dam. Since the post-implantation survival index was normal, the lower number of pups per litter was considered not a developmental effect. After birth, the body weights of pups were similar in all groups. During lactation, retardation of growth of the pups from litters at 500 mg/kg bw/day was observed resulting in lower body weights of these pups on PND 13 of approximately 11% in comparison with the other groups. No further treatment-related changes were noted in the other developmental parameters

investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, macroscopy, anogenital distance, nipple retention and thyroid hormone assessment).

In conclusion, treatment with Phytantriol by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg bw/day revealed parental toxicity at 500 mg/kg bw/day, expressed as reduced body weight gain and food consumption. Furthermore, non-adverse increases in enzyme levels of ALAT and ASAT and liver weights (both sexes), hepatocellular hypertrophy (females only) and mild local reaction in the stomach to the test item in males (squamous cell hyperplasia and edema) were observed. Treatment-related effects in reproduction were observed as reduced number of implantation sites at 150 and 500 mg/kg bw/day, being statistically significant only at 500 mg/kg bw/day. A corresponding reduction of mean number of living pups per litter was observed at 500 mg/kg bw/day only. Retardation of growth of pups during lactation was observed at 500 mg/kg bw/day. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 150 mg/kg bw/day

Reproduction NOAEL: 150 mg/kg bw/day

Developmental NOAEL: 150 mg/kg bw/day