Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 April 2019 to 18 July 2019 (experimental dates)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The animals were treated with the test item formulation or vehicle on 7 days per week between GD 5 and GD 19, except for animals no. 10, 11 (C group), 35, 36 (LD group) and 60 (MD group), which were treated from GD 7 to GD 19. Inadvertently, start of treatment was delayed for dams showing E+ vaginal smear on 25 May 2019. In principle the missed administrations might have masked a test item related effect on early development of the fetuses. However, considering the low number of animals affected (2/24 controls; 2/25 LD group animals and 1/25 MD group animals) and the fact that all HD animals were correctly dosed, the validity of the study is not assumed to be affected. This is supported by the fact that the HD level marks the NOAEL in this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Deviations:
Inadvertently, start of treatment was delayed for dams showing E+ vaginal smear on 25 May 2019.
Impact:
In principle the missed administrations might have masked a test item related effect on early development of the fetuses. However, considering the low number of animals affected (2/24 controls; 2/25 LD group animals and 1/25 MD group animals) and the fact that all HD animals were correctly dosed, the validity of the study is not assumed to be affected. This is supported by the fact that the HD level marks the NOAEL in this study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test substance:
- Name of test material (as cited in study report): 3-Methyl-1,5-Pentanediol
- Molecular weight: 118.20
- Physical state: Colorless liquid
- Analytical purity: 99.18%
- Lot/batch No.: 63136
- Expiration date of the lot/batch:
- Storage condition of test material: Nitrogen substituted, and stored sealed at room temperature in a dark place
- Other: Specific gravity: 0.97 (20°20); Boiling point: 270°C; Freezing point: Less than -50°C; Manufacturer: Kuraray Co.; Supplier: Ministry of Health and Welfare Environmental Health Bureau Planning Section Office for Environmental Chemicals Safety; Date of receipt: 1995-09-6;

Medium:
-Name: Japanese Pharmacopoeia water for injection (Otsuka Pharmaceutical Factory Co.,)
-Lot No. 5C82 and 5K81
-Storage: At room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Rat
- Strain: Wistar
- Age at dosing: 13-14 wks
- Weight at dosing: Females: 229-288 g
- Source: Charles River, Germany
- Acclimation period: 5 days
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Municipal water, ad libitum (pH adjusted to pH 2-8)
- Housing: Individually housed, except during cohabitation period with 2 females paired with 1 male and during pre-mating where females were kept in groups of 2

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ±3°C
- Humidity: 55 ± 10%
- Air changes: ca. 10/h
- Photoperiod: 12 h light/dark

In life dates: 2 April 2019 to 18 July 2019 (experimental dates))

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad injectionem
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
Vehicle and/or positive control: Water for injection (aqua ad injectionem) / n.a
- Concentration in vehicle: The test article was prepared at concentrations of 0, 20, 60 and 200 mg/mL. The prepared test formulations were prepared fresh on each day of dosing, under magnetic stirring during dosing.
- Amount of vehicle (if gavage): dose volume: 5 mL/kg bw
- Lot/batch no. (if required): 806148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test article was prepared at concentrations of 0, 20, 60, and 200 mg/mL. A separate study (Eurofins Munich Study No. 187545) concluded that test article concentrations of 15 and 250 mg/mL were stable for =10 days at room temperature, refrigerated (2-8°C) and frozen (-15 to -35°C). These samples were homogenous after 60 minutes (no stirring). The prepared test formulations were prepared and stored in conjunction with the previous stability data generated.
Verification and homogeneity of the test article formulations preparation containing were determined by the analysis of three samples (from upper, middle, lower strata) from each dose level prepared at the start and end of the dosing phase.

Acceptance criteria for concentration analysis:
- Mean concentration of test article formulation 90-110% of nominal

Stability analysis:
- Stability of test article had previously been verified.
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperm were observed in the vaginal smear was considered as GD ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number. After achieving 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
from gestation day (GD) 5 through to GD 19, with scheduled necropsy on GD 20
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 or 25/gp
Control animals:
yes, concurrent vehicle
Details on study design:
After an acclimatisation period of ca. 5 days, virgin female rats were paired 1 male to 2 females with breeder rats of the same source. Female rats with spermatozoa observed in a smear of the vaginal contents observed in situ were considered to be day 0 of gestation. Animals were assigned randomly to dose groups on the basis of gestation day (GD) 0 body weights, with each group consisting of 24 or 25 females /group, thereby ensuring 20 litters were available for analysis at termination.

The dose levels were selected based on the results from a range-finding study (study number: 187542). In the dose-range developmental toxicity study, Wistar rats were administered 3-methylpentane-1,5-diol on GD 5 or 6 - 19 at dosages of 0, 100, 300 and 1000 mg/kg bw/d. All rats survived to scheduled euthanasia; and, there were no clinical or necropsy observations related to administration of the test article at the highest dosage level tested. Body weights were comparable among the groups. There were no marked differences among the groups. Absolute and relative food consumption values were comparable among the groups. Dosages as high as 1000 mg/kg bw/day of 3-methylpentane-1,5-diol did not affect any parameter evaluated at caesarean-sectioning. There were no marked differences among the groups. The litter averages for corpora lutea, implantations, live fetuses, late resorptions, fetal body weights, percent male fetuses and were comparable among the four dosage groups There were no dead fetuses or late resorptions and no dam had a litter consisting of only resorptions. All placentae appeared normal.

An increase in total resorptions was observed at the mid and high dose gps which exceeded the concurrent vehicle control and/or the historical control range. A group total of 12 resorptions were observed at the mid dose group, with all 12 attributed to early resorption. In the high dose group, a group total of 8 resorptions were observed.

It was recognized that a shift in the number of dams with post implantation loss occurred through the dose groups. No conclusion on a dose related effect could be made due to the low number of animals dosed. However it should be noted that there was no dose dependency in either pre or post implantation loss, with the group values consistent with the historical control range (-/+2 SD). However one vehicle control animal (#7: 81.82%) exceeded the historical control range -/+2 SD range (20.61 – 46.76) for pre-implantation loss (20.61 – 46.76%), with one animal from the mid (#19: 50.00%) and one animal from the high dose (#28: 33.33%) exceeding the historical control range (-/+2 SD: 18.85 – 32.70%).

Based on these data, dosages of 0, 100, 300 and 1000 mg/kg bw/day of 3-methylpentane-1,5-diol were recommended for the developmental toxicity study in rats, employing a dose volume of 5 mL/kg bw. The 1000 mg/kg bw/day dosage was expected to be a NOAEL for both maternal and embryo-fetal toxicity.

Examinations

Maternal examinations:
- Observations: The animals were checked for mortality at least once daily. The rats were observed for general appearance at least once daily.
- Body weights: Recorded at least once during the acclimation period, on GD 0, 5, 8, 11, 14, 17 and 20. (prior to necropsy).
- Food consumption: Recorded on GD 0, 5, 8, 11, 14, 17 and 20
- Water consumption: Not conducted
- Ophthalmological examination: Not conducted
- Mating performance: Evaluated daily during the cohabitation period. Dams were sacrificed on day 20 of gestation.
- Haematology and clinical chemistry: Thyroid hormones levels from all dams were assessed at the end of the treatment prior. At termination, blood samples were collected and assessed for serum levels for thyroid hormones (T3, T4 and TSH).
- Urinalysis: Not conducted
- Organ weights: Gravid uterus, thyroid gland
- Histopathology: limited to the thyroid/parathyroid gland
Ovaries and uterine content:
- Ovarian and uterine examinations: the gravid uterus was weighed, uterus opened and the contents were examined. The fetuses were removed from the uterus and placed in individual containers (or a tray). The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses, and early and late resorptions. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Non-responding term fetuses were considered to be dead. Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption. Uteri of apparently non-pregnant rats were examined by staining with 10% ammonium sulfide to confirm the absence of implantation sites.

- Necropsy: rats were subjected to a gross necropsy examination, which included an evaluation of the thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Gross lesions were collected for all animals. Representative samples of the tissues (cervix, collected with uterus: including non-pregnant animals; gravid uterus, all animals; gross lesions, all animals; liver, all animals; ovaries, including all non-pregnant animals; uterus, including all non-pregnant animals) were collected and preserved in 10% neutral buffered formalin. Thyroid and gravid uterus were weighed. The thyroid and gross lesions were examined histopathologically on all animals.
Fetal examinations:
Fetuses were euthanized via an intraperitoneal injection of sodium pentobarbital (first 20 dams/gp analysed).

- Visceral examination: Approximately one-half of the fetuses in each litter were examined using a modification of the micro-dissection technique of Staples (1964). Each fetus was fixed in Bouin's solution and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The abdominal and thoracic cavities of all fetuses were dissected and examined for visceral anomalies. The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off centre and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect. The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus.

Craniofacial examination: A single foetus was decapitated and the head of the foetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.

The decapitated carcasses were not retained.

- Skeletal examination: The remaining fetuses (approximately one-half of the fetuses in each litter) were examined after staining with Alcian Blue and Alizarin red staining technique. Following examination, skeletal preparations were retained in glycerin, with thymol added as a preservative. The skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centres. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centres and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus. archived with the raw data.
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed animals with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).
Indices:
% pre-implantation loss = (No. of corpora lutea – No. of implantations) / no. of corpora lutea x 100
% post implantation loss = (No. of implantations - no. of live fetuses) / No. of implantations x 100
Pregnancy rate= No. of pregnancies / No. of females mated or sperm positive x 100
Historical control data:
Yes (refer below)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test article related effects were observed.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
n/a
Mortality:
no mortality observed
Description (incidence):
All animals survived until the date of scheduled sacrifice
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight remained unaffected by treatment with the test article and increased with the progress of the study in the control and all test article treated groups throughout the study period. No statistical significance was achieved for body weight or body weight gain in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the control group (refer to Table 7.8.2/02-2).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of food consumption data revealed markedly lower group mean food consumption during gestation day 8-11 and day 17-20 in the high dose group when compared with the control although statistical significance was achieved only during gestation day 8-11.
Although there was reduction in group mean food consumption observed in the high dose group during gestation day 8-11 and 17-20, this effect could was attributed to very low individual food consumption value from 3/25 dams (#95, 96, 97) during GD 8-11 and dam #90 during GD 17-20. Individual food consumption values from all other high dose group dams were comparable with the controls. Since no effect on body weight effects were observed, this effect on food consumption during GD 5-8 and 17-20 in the high group was considered not to be toxicologically relevant (refer to Table 7.8.2/02-6)
Food efficiency:
not examined
Description (incidence and severity):
n/a
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test article related effects were observed.
Ophthalmological findings:
not examined
Description (incidence and severity):
n/a
Haematological findings:
not examined
Description (incidence and severity):
n/a
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test article related effects were observed in TSH, T3, T4 levels (refer to Tables 7.8.2/02-3)
Urinalysis findings:
not examined
Description (incidence and severity):
n/a
Behaviour (functional findings):
not examined
Description (incidence and severity):
n/a
Immunological findings:
not examined
Description (incidence and severity):
n/a
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test article related effects were observed on gravid uterus or thyroid weights (refer to Tables 7.8.2/02-2; 3, respectively)
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related gross abnormalities were observed
Neuropathological findings:
not examined
Description (incidence and severity):
n/a
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related effects on thyroid histopathology were observed, with the only finding limited to a single incidence of follicular c.hyperplasia in a single control animal. No other effects were observed
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
n/a
Other effects:
not examined
Description (incidence and severity):
n/a

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
It is recognized that a shift in the number of dams with post implantation loss occurred through the dose groups. No conclusion on a dose related effect can be made due to the low number of animals dosed. However it should be noted that there was no dose dependency in either pre or post implantation loss, with the group values consistent with the historical control range (-/+2 SD). However one vehicle control animal (#7: 81.82%) exceeded the historical control range -/+2 SD range (20.61 – 46.76) for pre-implantation loss (20.61 – 46.76%), with one animal from the mid (#19: 50.00%) and one animal from the high dose (#28: 33.33%) exceeding the historical control range (-/+2 SD: 18.85 – 32.70%) (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5)
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
An increase in total resorptions was observed at the mid and high dose gps which exceeded the concurrent vehicle control and/or the historical control range. A group total of 12 resorptions were observed at the mid dose group, with all 12 attributed to early resorption. This exceeded both the concurrent vehicle control group value (total of 2 resorptions [1 early / 1 late]) and historical control observed range (total upper value of 8 resorptions [with 0.0 - 8.0 for early / 0.0 - 1.0 for late), however individual values from all animals (including the 3 animals contributing to this elevated total gp value) were consistent with the observed range.

In the high dose group, a group total of 8 resorptions were observed. This exceeded the concurrent vehicle control group value (total of 2 resorptions [1 early / 1 late]) but were consistent with the historical control observed range (total upper value of 8 resorptions [with 0.0 - 8.0 for early / 0.0 - 1.0 for late), however individual values from all animals (total resorptions ranging from 0 - 3) were consistent with the observed range (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5)
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test article related effect on litter data (i.e. mean no. of corpora lutea, implantations, resorptions, pre- and post implantations and live young), at any dose level investigated. Sex ratio was also unaffected by treatment (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no test article related effect on litter data (i.e. mean no. of corpora lutea, implantations, resorptions, pre- and post implantations and live young), at any dose level investigated. Sex ratio was also unaffected by treatment (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Changes in pregnancy duration:
not examined
Description (incidence and severity):
n/a
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Successful mating resulted in 21/25 pregnancies in the low, 25/25 in the mid and 22/25 in the high dose group compared to 22/24 pregnancies in the control group. The variation in pregnancy rates of 91.67% in the control group compared to treatment groups (84% in LD, 100% in MD and 88% in HD) was not considered dose related, and therefore not deemed test article related (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5). Females which were sperm positive but did not produce a litter were excluded from subsequent calculations.
Other effects:
not examined
Details on maternal toxic effects:
No evidence of maternal toxicity was observed up to 1000 mg/kg bw/day, the maximum recommended dose for sub-acute repeat dose studies

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test article-related effects were observed for litter data including group mean fetus weight, total number of fetuses, number of males and females, total litters, male and female litter weight (refer to Tables 7.8.2/02-4 and Table 7.8.2/02-5).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect on sex ratio was observed (refer to Tables 7.8.2/02-4 and 7.8.2/02-5)
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test article-related effects were observed for litter data including group total litter size, male and female fetal weight (refer to Tables 7.8.2/02-4 and 7.8.2/02-5)
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test article related external effects were observed.

Low incidences of umbilical hernia (1 in in control), short tail (1 in the high dose) and skin discolouration due to injection of sodium pentobarbital (Narcoren) (1 each in low dose and high dose) were noted in isolated fetuses of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single fetuses, they were considered to be incidental in nature and unrelated to treatment (refer to Table 7.8.2/02-7).

No effect on anogenital distance was observed (refer to Tables 7.8.2/02-4 and 7.8.2/02-5).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination: no test article related skeletal effects were observed (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

Rudimentary/short ribs are considered as transient abnormalities. Furthermore, rudimentary/short ribs finding values were within the upper limit of historical control data range and therefore considered not to be treatment-related but spontaneous in nature (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

The finding of a caudal shift of the pelvic girdle (left) in HD group was well within historical control data range (22.22 %) and changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain and therefore this finding was considered not to be adverse (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

There was no statistical significance and no indication of a test item-related trend in the type and/or litter incidences of other skeletal findings and they were therefore considered to be spontaneous in nature. Frequencies for litter incidences of few skeletal findings were even less in the treated groups compared to the controls. Therefore, these findings are considered not to be treatment-related but rather solely spontaneous in nature.

Fetal ossification site averages: no fetal ossification effects were observed (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification of skull –basioccipital (20 % compared to 15% in controls), 5th sternebra (80% compared to 55% in controls), femur (15% compared to 0% in controls), left pelvic girdle- caudal shift (15% compared to 10% in controls), right 7th cervical rib (15% compared to 5% in controls) and rudimentary left 14th rib (65% compared to 50% in controls) were observed in the high dose group when compared to the control group (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).

The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the high dose group were either marginally higher or within historical control data range. Generally delayed ossification might be correlated to the lower pup weight observed in the high dose group, and is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is considered not to be adverse (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test article related visceral effects were observed.

Statistical analysis of visceral data revealed statistically significantly higher group litter incidences for bilateral azygos vein and long thymus in MD group when compared with the controls. Due to lack of dose dependency and consistency, these findings were considered not toxicologically relevant.

There were also higher litter incidences, but without achieving statistical significance for supernumerary liver lobe and dilated renal pelvis observed in treatment groups. However, values were well within upper limit of historical control data range so that no toxicological relevance was attributed to it. Dilated renal pelvis is a common finding in rodent studies and is classified as a variation as this is transient and likely to be postnatally reversible (refer to Tables 7.8.2/02-7 and (refer to Table 7.8.2/02-8).
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination: a few predominant findings (red material in perimeningeal space and increased perimeningeal space) at low frequencies generally comparable to or in some cases slightly higher in frequency in the dose groups compared to the controls. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for litter incidence of any of these findings.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

Table 7.8.2/02-1: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: dose formulation analysis

Parameters

Dose (mg/mL)

20

60

200

Week 1

98.2

93.9

98.6

Week 3

104.8

98.4

102.4

Table 7.8.2/02-2: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: body weight effects

Parameters

¿ (mg/kg bw/d)

0

100

300

1000

No. of animals treated

24

25

25

25

Body wt (g)

Day
0
5
20


239.0 ±8.6
256.6 ±9.8
341.7 ±19.5


244.1 ±16.0
260.4 ±13.2
348.6 ±21.7


238.6 ±14.2
253.8 ±11.3
341.5 ±17.2


237.5 ±10.5
254.7 ±9.0
334.0 ±21.5

Gravid uterine wt (g)


58.11 ±13.84


64.49 ±15.96


60.90 ±9.65


56.97 ±12.89

Corrected body wt (g)

Day
20a


283.61 ±12.19


284.09 ±15.52


280.58 ±15.02


276.99 ±16.29

Body wt gain (g)

Days
0-20a


44.61 ±10.28


39.94 ±11.46


42.02 ±10.54


39.44 ±14.90

a gestational body weight – gravid uterine weight

Table 7.8.2/02-3: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: thyroid weights and hormone levels

Parameters

Dose group (mg/kg bw/d)

 

0

100

300

1000

 

No. of animals treated

24

25

25

25

 

Terminal bwt (g)

341.73 ±19.51

348.57 ±21.71

341.48 ±17.17

333.95 ±21.50

 

Thyroid

Abs (g)
Rel. (g%)

0.0291 ±0.0072
0.0085 ±0.0020

0.0260 ±0.0055
0.0075 ±0.0016

0.0262 ±0.0048
0.0077 ±0.0014

0.0259 ±0.0042
0.0077 ±0.0011

 

T3 (pg/mL)

2648.40 ±769.91

2654.21 ±475.05

2694.03 ±660.18

2882.47 ±644.51

 

T4 (nmol/L)

43.30 ±7.32

42.00 ±10.72

46.04 ±7.46

45.64 ±6.70

 

TSH (pg/mL)

2235.61 ±965.87

2130.23 ±723.71

2083.16 ±620.52

2337.24 ±946.27

 

Abs.: absolute
Rel.: relative to body weight

 

Table 7.8.2/02-4: Overview of developmental toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: selected caesarean section data

Parameters

Dams (mg/kg bw/d)

0

100

300

1000

No. of animals mated

24

25

25

24

Animals pregnant

24

25

25

25

Non-pregnant

0

0

0

0

No. of animals used for calculation

22

21

25

22

Maternal wastage

n/a

n/a

n/a

n/a

Total corpora lutea
[/dam]

281
12.8 ±1.5

276
13.1 ±2.0

322
12.9 ±1.9

260
11.8 ±2.4

Total implantations
[/dam]

252
11.5 ±3.2

256
12.2 ±2.4

303
12.1 ±1.4

245
11.1 ±2.8

Total no. of litters

22

21

25

22

Total live fetus
[/dam]

234 (100%)
[10.6 ±3.0]

238 (100%)
[11.3 ±2.6]

275 (100%)
[11.0 ±1.7]

230 (99.6%)
[10.5 ±2.9]

Total dead fetus

0.0 (0%)

0.0 (0%)

0.0 (0%)

1.0 ±0.2 (0.4%)

Total resorptions
[early/late]

18
[18/0]

18
[18/0]

28
[28/0]

15
[15/0]

Resorptions/dam
[early/late]

0.8 ±1.3
[0.8 ±1.3 / 0.0]

0.9 ±1.1
[0.9 ±1.1 / 0.0]

1.1 ±1.4
[1.1 ±1.4 / 0.0]

0.7 ±0.8
[0.7 ±0.8 / 0.0]

Abortion sites

0

0

0

0

 

M

F

M

F

M

F

M

F

Litters used for calculation

22

21

25

22

Fetal wt (g)

3.9 ±0.4

3.7 ±0.4

4.1 ±0.4

3.9 ±0.5

3.9 ±0.3

3.7 ±0.2

4.0 ±0.4

3.8 ±0.4

Mean fetal wt (g)

3.8 ±0.4

4.0 ±0.5

3.8 ±0.2

3.9 ±0.4

Anogenital distance

- Cube root of fetus wt (g)

1.56 ±0.05

1.57 ±0.07

1.60 ±0.07***

1.56 ±0.07**

1.57 ±0.05

1.55 ±0.04

1.58 ±0.05

1.55 ±0.05

- AGD (mm)

2.67 ±0.38

1.41 ±0.30

2.84 ±0.40**

1.55 ±0.32**

2.81 ±0.38*

1.51 ±0.36*

2.85 ±0.35**

1.49 ±0.24

- Relative AGD

1.71 ±0.23

0.92 ±0.19

1.78 ±0.24

0.99 ±0.21*

1.79 ±0.24*/

0.98 ±0.22*

1.81 ±1.81*

0.97 ±0.16

Sex ratio (%¿)

54.0 ±24.6

54.9 ±15.8

51.1 ±18.1

43.2 ±18.9

Implantation loss
(pre-/post-) [%]

1.3 ±2.3 / 0.8 ±1.3
[11.2 ±21.1/6.2 ±9.8]

1.0 ±1.9 / 0.9 ±1.1
[6.8 ±14.3/7.4 ±9.1]

0.8 ±0.7 / 1.1 ±1.4
[4.9 ±10.5/9.1±11.1]

0.7 ±1.4 / 0.7 ±0.8
[5.9 ±13.6/7.0 ±7.6]

Malformed fetuses/litter

0/22

0/21

0/25

0/22

*p=0.05; **p=0.01

Incidence of prenatal events in rats treated orally (via gavage) with 3 -methylpentane-1,5 -diol

No of Dams affected

Dose group (mg/kg bw/day)

 

0

100

300

1000

Abortions

0

0

0

0

Early deliveries

Not reported

Stillbirths

Not reported

Resorptions (early/late)

11

(11/0)

12

(12/0)

14

(14/0)

11

(11/0)

Dead Fetus

0

0

0

1

 

Table 7.8.2/02-5: Laboratory historical control data (2011-2019) Wistar rats

Parameters

n

Observed range

Mean ±SD

Mean -/+2SD

Uterine data

Uterus weight

848

0.5 – 109.5

60.60 ±14.80

31.01 – 90.20

Corpora lutea/dam

851

1.0 – 23.0

13.11 ±2.07

8.96 – 17.26

Implantations/dam

851

1.0 – 18.0

11.58 ±2.74

6.10 – 17.06

Sex ratio

835

0.0 – 11.0

1.25 ±1.10

-0.96 – 3.45

Total live fetus

849

0.0 – 17.0

10.86 ±2.84

5.18 – 16.54

Total dead fetus

848

0.0 -1.0

0.00 ±0.06

-0.12 ±0.12

Total resorptions
[early/late]

849

0.0 – 11.0
[0.0 – 9.0 / 0.0 – 11.0]

0.73 ±1.16
[0.69 ±1.09 / 0.04 ±0.41]

-1.59 – 3.04
[-1.49 – 2.86 / -0.78 – 0.86]

No. of implantations

851

1.0 – 18.0

11.58 ±2.74

6.10 – 17.06

Pre-implantation loss (%)

851

0.0 – 90.9

12.01 ±16.59

-21.17 – 45.19

Post-implantation loss (%)

849

0.0 – 100.0

6.89 ±12.41

-17.93 – 31.71

Mean litter weight data (g)

Mean fetal weight

845

2.12 – 5.69

3.65 ±0.43

2.80 – 4.50

Total litter weight

845

3.60 – 85.30

39.44 ±10.17

19.09 – 59.79

Male litter data

845

0.00 – 58.30

20.20 ±8.38

3.44 – 36.97

Female litter data

845

0.00 – 44.20

19.24 ±7.50

4.24 – 34.23

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw/day (the maximum recommended dose for sub-acute repeat dose studies in accordance with current guideline requirements) based on no adverse effects observed up to the highest dose tested.
Executive summary:

In a developmental toxicity study 3-methylpentane-1,5-diolwas administered to 24 or 25 Wistar female rats/groupby oral gavageonce daily at dose levels of 0 (water for injection), 100, 300 or 1000 mg/kg bw/day from days 5 through 19 of gestation, employing a dose volume of 5 mL/kg bw. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ovarian and uterine examinations, fetal examinations, gross necropsy findings, thyroid hormone assessment in dams and histopathology.

All rats survived to scheduled euthanasia. There were no clinical observations considered related to administration of 3-methylpentane-1,5-diol. Body weights and body weight gains were not affected by dosages of 3-methylpentane-1,5-diol as high as 1000 mg/kg bw/day. Absolute and relative food consumption values were comparable for all intervals evaluated except days 8-11 and 17-20 in the high dose group when compared with the control although statistical significance was achieved only during gestation day 8-11. Although there was reduction in group mean food consumption observed in the high dose group during gestation day 8-11 and 17-20, this effect could was attributed to very low individual food consumption value from 3/25 dams (#95, 96, 97) during GD 8-11 and dam #90 during GD 17-20. Individual food consumption values from all other high dose group dams were comparable with the controls. Since no effect on body weight effects were observed, this effect on food consumption during GD 5-8 and 17-20 in the high group was considered not to be toxicologically relevant.

Pregnancy occurred in 24, 25,25, and 25 rats in the 0, 100, 300 and 1000 mg/kg bw/day dosage groups, respectively. Dosages as high as 1000 mg/kg bw/day of 3 -methylpentane-1,5-diol did not affect any parameter evaluated at caesarean-sectioning.

No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of the test article as high as 1000 mg/kg bw/dose. There were no dosage-dependent or significant differences in the litter or fetal incidences of any gross external, soft tissue or skeletal alterations. There were no biologically-important differences in ossification site averages among the four dosage groups

Under the conditions of this study, the NOAEL for both maternal and developmental toxicity was 1000 mg/kg bw/day (the maximum recommended dose for sub-acute repeat dose studies in accordance with current guideline requirements) based on no adverse effects observed up to the highest dose tested.