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Diss Factsheets

Administrative data

Endpoint:
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:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
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:
other: 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 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-treatment: 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 were 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.
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.
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.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Females were housed in Macrolon plastic cages (MIII type, height 18 cm).
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

Maternal examinations:
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 th
e 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.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability
The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs
At least once daily, detailed clinical observations were made for all animals.
Body weights
Live pups were weighed on PND 1, 4, 7 and 13.
Sex
Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.
Anogenital distance
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention
On PND 13, all males in each litter were examined for the number of areola/nipples.
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 in tergroup 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.
Indices:
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: maternal animals

General toxicity (maternal animals)

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:
no mortality observed
Description (incidence):
No mortality was observed for females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In females treated at 500 mg/kg bw/day, body weights and body weight gain were 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):
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 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 female rats were considered not to be affected after treatment up to and including 500 mg/kg bw/day. Any statistically significant changes for 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 female rats at 500 mg/kg bw/day, achieving levels of statistical significance for ALAT in both sexes when compared to controls.
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.
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 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. 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
Description (incidence and severity):
Increased mean relative liver weights were observed in males and females at 500 mg/kg bw/day, of approximately 9% when compared to controls, achieving a level of statistical significance in males only. Due to the low magnitude of increase, all liver weights remained well within normal limits for both sexes.
Mean weights of several other organs, either absolute weights or relative to body weight, in male and female rats at 500 mg/kg bw/day were (statistically significantly) different from controls. For these organs these differences were the result of the accompanying lower (terminal) body weight in these high dose animals in comparison with controls. The concerning changes included the absolute weights of the heart (both sexes) and the adrenals and seminal vesicles in males and uterus in females, and the relative weights of the brain (both sexes) and testes in males. The observed changes in these organs were considered not to be toxicologically relevant, also taking into account that no histopathological findings were observed in these organs.
Organ weights and organ to body weight ratios in animals treated at 50 and 150 mg/kg bw/day were similar to control levels.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment in females.
Neuropathological findings:
not examined
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 liver of the 500 mg/kg/bw day females (minimal/slight hepatocellular hypertrophy).
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

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A dose related decrease in the number of implantation sites was apparent, being statistically significantly lower than controls in females at 500 mg/kg bw/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 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.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
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 dam from the control group 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.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. The event that one out of eight pregnant females at 150 mg/kg bw/day and one out of nine at 500 mg/kg bw/day had implantation sites only (and thus did not give birth to living offspring) was considered to have occurred by chance and of no toxicological significance.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. The event that one out of eight pregnant females at 150 mg/kg bw/day and one out of nine at 500 mg/kg bw/day had implantation sites only (and thus did not give birth to living offspring) was considered to have occurred by chance and of no toxicological significance.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body 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.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
effects 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.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
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.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to
treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
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.
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.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
yes

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 SPF bred 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) and hepatocellular hypertrophy (females only) 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