Administrative data
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Attached justification
Cross-reference
Data source
Materials and methods
Test guideline
#1 - Test guideline
adopted 29 July 2016
Test material
Test item name as stated in the report: 3-methyl-1,3-butanediol
Alternate Identification: Isopentyldiol
SOURCE OF TEST MATERIAL
- Source: Kuraray Co., Ltd., Otemachi, Chiyoda-ku, Tokyo (Japan)
- Sponsor and lot/batch number of test material: 23515
- Purity (GC): ≥99.9 %
- Expiration Date: 02 December 2023
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature protected from light, humidity and under nitrogen purge.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability analyses performed previously in conjunction with Study No. 20384993 demonstrated that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study (In a concentration range of 10–120 mg/mL for at least 8 days at room temperature and +4 °C and for at least 3 weeks at -20 °C).
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Dosing formulations were prepared at appropriate concentrations in purified water as vehicle to meet dose level requirements according to standard operating procedures of the Test Facility.
Test animals
Wistar Han IGS
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
TEST ANIMALS
- Source: Charles River Laboratories France, 327 Impasse du Domaine Rozier, Les Oncins, 69210 Saint-Germain-Nuelles, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 10 weeks, females: 11 weeks
- Weight at study initiation: Males: 315 - 368 g; Females: 187 - 261 g
- Fasting period before study: no
- Housing: group housed during pre-mating period (up to 4 for males and 5 females), 1 male and 1 female housed together during mating, after mating males were group-housed (up to 4) and females single housed with litter up to lactation period in plastic cage containing appropriate bedding. For psychological/environmental enrichment, animals were provided with items such as a wooden gnaw block and shredded paper, except when interrupted by study procedures/activities. It was considered that there were no known contaminants in the enrichment that could have interfered with the outcome of the study.
- Diet: ad libitum, complete rodent diet (Reference No. SM-VRF1-C12), sterilized by irradiation
- Water: ad libitum, softened and filtered (0.2 µm) municipal drinking water.
- Acclimation period: 21 days and 7 days before the start of dosing for females and males, respectively.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): ≥ 35
- Air changes (per hr): ≥ 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2022-11-07 To: 2023-01-02
Administration / exposure
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OTEC H2O sterile water for technical use
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at appropriate concentrations to meet dose level requirements according to standard operating procedures of the Test Facility
VEHICLE
- Amount of vehicle (if gavage): 10 mL
- Lot/batch no. (if required): C0822
- M/F ratio per cage: 1/1
- Length of cohabitation: for a maximum of 14 days
- Proof of pregnancy: presence of sperm in a vaginal smear or a vaginal plug referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another male with proven fertility/Further matings after two unsuccessful attempts: Not necessary, all pairs of rats mated within the designated mating period.
- After successful mating each pregnant female was caged (how): single
Analyses were performed by gas chromatography (GC) method using a validated analytical procedure (Test Facility Study No. 20384993).
All analyzed samples for formulations prepared at nominal concentrations of 12, 35 and 100 mg/mL of 3-methyl-1, 3-butanediol in vehicle "purified water" taken from each preparation used for dosing on the first day of treatment were within the acceptance criteria (± 10 %). Furthermore the test item prepared as a solution in the vehicle was homogenous as the RSD on the six aliquots (Top, Middle, Bottom) was ≤ 1 %. Finally, no test item was present in the vehicle sample.
Males: from 14 days before mating, throughout mating and up to the day before necropsy (i.e., 35 days)
Females: from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy (i.e., LD 13-15).
Daily
Doses / concentrations
#1 - Doses / concentrations
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#2 - Doses / concentrations
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#3 - Doses / concentrations
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#4 - Doses / concentrations
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10/sex/dose
- yes, concurrent vehicle
- Dose selection rationale: The dose levels (0, 120, 350, and 1000 mg/kg/day) were selected in the present study in conjunction with the Sponsor, based on a 28-day oral toxicity study according to OECD guideline 407. In this study, dose levels of 0, 15, 150, and 1000 mg/kg/day were administered by daily oral gavage to Wistar rats. No treatment related effects were observed up to the highest dose level. A no observed adverse effect level (NOAEL) of 1000 mg/kg/day was determined in this study.
- Fasting period before blood sampling for clinical biochemistry: no
no
Examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and at least once after dosing. At least once daily on non-dosing days (The pre-dose clinical observation was not performed on Day 16 for all animals and on Day 23 for Male Nos. 1001 to 1007, in error. As no clinical signs were observed after dosing, this deviation was considered to have had no impact on the study outcome.)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed on each weighing day
BODY WEIGHT: Yes
- Time schedule for examinations: All females: weekly during pre-mating and mating (only pre-mating data are reported), then on GD0, GD4, GD7, GD11, GD14, GD17, GD20,LD1, LD4, LD7, and LD13. All males: once pretest and then weekly from Day 1
FOOD CONSUMPTION
All females: Weekly during pre-mating (from Day 1), then on GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17 and 17 to 20, and on LD1 to 4, 4 to 7 and 7 to 13 (On 06 Dec 2022, the food consumption values of all females were found to be negative, most probably following a food distribution error without associated data recording. As a consequence, these aberrant values were excluded from the mean calculations and statistical analysis for the periods GD7 to GD11 or GD11 to GD14 and GD0 to GD20. This loss of data had no impact on the study outcome since there was no change in food consumption that affected body weight gain in any group).
All males: Weekly during premating (from Day 1)
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OTHER:
Blood samples (0.6 mL plasma in K3EDTA) for T4 thyroid hormone analyses were collected from F0 males after 35 days of treatment from jugular vein without anesthesia or sublingual vein under slight isoflurane anesthesia. Samples were stored on wet ice and centrifuged within 60 minutes at 1800 g for 10 minutes at approximately 4 °C before freezing at -70 °C/-90 °C within 120 minutes after blood collection.
Estrous cycles: daily from 2 weeks before treatment, the first 2 weeks of treatment and until identification of mating or separation from the male. Vaginal smear taken on the day of scheduled necropsy.
Parameters examined in male parental generation of Groups 1 (control) and 4 (high dose) and for macroscopic lesions from all dose groups:
testis weight, epididymis weight, For the testes from all adult males of Groups 1 (control) and 4 (high dose) and all males suspected to be infertile, in addition to the haematoxylin and eosin-stained slide, an additional slide was prepared and examined, stained with PAS. A detailed, qualitative evaluation of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings was recorded, where appropriate.
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (random selection of 4/sex/litter, if possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups born (live and dead), external abnormalities of the pups, number, weight and sex of pups alive on PND1, 4, 7, 10 and 13, anogenital Distance (AGD) was measured on PND1 and normalized to the cube root of body weight. All males in each litter were examined for the number of areola/nipples on PND13.
Blood samples were collected from at least 2 of the surplus pups (if possible from 1 male and 1 female pup) from all litters culled on PND4 and 2 pups per litter where possible on PND13 or 15 used for determination of serum T4 and possibly TSH levels.
GROSS EXAMINATION OF DEAD PUPS: yes. Pups (including those found dead) were necropsied and submitted to a full external macroscopic examination and examination of the abdominal and thoracic cavities. Any
abnormalities observed were recorded but not preserved. For pups found dead, the stomach was examined and the absence of milk was recorded if applicable, and defects or cause of death were evaluated if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs.
OTHER
Blood samples (0.6 ml plasma in K3EDTA) for T4 thyroid hormone analyses were obtained from 2 pups/litter (1 male and 1 female) at PND 13 or 15 by intracardiac injection. Samples were stored on wet ice and centrifuged within 60 minutes at 1800 g for 10 minutes at approximately 4° C before freezing at -70° C/-90° C within 120 minutes after blood collection.
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals
GROSS NECROPSY
- All animals (including the female with total litter death) were submitted to necropsy procedures including an examination of the following: external surface, all orifices, cranial cavity, thoracic and abdominal cavities and organs and their contents, the carcass.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 in section "Any other information on materials and methods incl. tables" were prepared for microscopic examination and weighed, respectively.
SACRIFICE/GROSS NECROPSY
- The F1 offspring not selected were sacrificed at 4 days of age (PND 4).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Full external macroscopic examination and examination of the abdominal and thoracic cavities. Any abnormalities observed were recorded but not preserved. For pups found dead, the stomach was examined and the absence of milk was recorded if applicable, and defects or cause of death were evaluated if possible. Each pup was sexed and examined for external defects with special attention being paid to
the external reproductive organs.
HISTOPATHOLOGY / ORGAN WEIGTHS: no
Descriptive Statistical Analyses:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported, as appropriate by dataset.
Inferential Statistical Methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and were reported at the 1% and 5% levels, unless otherwise noted.The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses were performed according to the matrix shown in table 2 below in section "Any other information on materials and methods incl. tables" when possible, but excluded any group with less than 3 observations.
Parametric/Non-Parametric:
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Non-Parametric:
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
Incidence:
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
The following reproductive indices were included Pre-Coital Interval (Days): (Sum of days until successful insemination/Number of inseminated females) *100 Female Mating Index (%): (Number of females with evidence of mating/ Number of paired females) *100 Female Fertility Index (%): (Number of pregnant females/ Number of females with evidence of mating) *100 Gestation Index (%): (Number of females with live pups/ Number of pregnant females) *100
Post Implant Loss/Litter (%): ((Number of implantations-Number of pups born)/Number of implantations)*100 Live Birth Index (%) :(Number of pups born alive/ Number of pups born) *100 Viability Index (%) :(Number of pups alive on PND4/ Number of pups alive at birth) *100 Lactation Index (%): Number of pups alive on PND 13/ Number of pups alive on PND 4)*100 Sex ratio (%): (Number of live male pups/ Total number of live pups)*100
Model and software
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Any other information on materials and methods incl. tables
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
There was no test item-related clinical sign in any group for either sex.
Clinical signs such as salivation, slight locomotor stereotypy, bent tail, scabs, thin fur cover or stained fur, and/or soft feces were noted sporadically across groups (including control) and were considered incidental.
There was no test item-related death in any group.
One female from the 120 mg/kg/day group was euthanized on LD1 following total litter death.
There was no test item-related effect on mean body weight gain and mean body weight throughout the dosing period for the males and during the premating, gestation and lactation periods for the females in any group (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
There was no test item-related effect on mean food consumption during the premating period in any group for either sex.
There was no obvious test item-related effect on mean food consumption during the gestation and lactation periods in any group for the females.
Minor variations were observed during the gestation period. Despite some differences that attained statistical significance, there was no trend or correlated data that suggested that these variations were test item-related.
There was no test item-related effect on mean T4 concentration in adult males (for more information, please refer to section "Any other information on results inlc. tables").
There were no test item-related effects on thyroid hormones in male rats, nor any other effects with regard to endocrine-related mode of actions on estrous cycles in female rats or organ weights and histopathology in adult rats of any dose group.
No test item-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to treatment.
Reproductive function / performance (P0)
There was no test item-related effect on estrous cyclicity in any group.
Mean length and regularity of the estrous cycles were comparable in all groups during the acclimatization and treatment periods (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
All pairs of rats mated in the control, 120, 350 and 1000 mg/kg/day groups. The mating index was therefore 100% in all groups.
All mated females were inseminated within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore comparable (less than 4 days) in all groups.
All mated females became pregnant in all groups except 1 in the 120 mg/kg/day group. This isolated case in the low dose group was incidental.
All pregnant females in the control, 120, 350, and 1000 mg/kg/day groups successfully completed delivery of liveborn pups. The mean duration of gestation was comparable in all groups (21.8 to 21.9 days) (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
Details on results (P0)
Effect levels (P0)
#1
- other: No adverse effects were observed
Target system / organ toxicity (P0)
#1
Results: P1 (second parental generation)
General toxicity (P1)
Reproductive function / performance (P1)
Details on results (P1)
Effect levels (P1)
Target system / organ toxicity (P1)
Results: F1 generation
General toxicity (F1)
There was no pattern in the incidence of pup clinical observations that suggested any association with maternal treatment.
Consistent with the total litter loss of one female from the 120 mg/kg/day group, there was a higher incidence of pups with no milk band present in the 120 mg/kg/day group compared with the control group.
There was no effect of maternal treatment on pup viability and litter size.
The live birth and viability indices (88.7 % and 87.2 %, respectively) in the 120 mg/kg/day group were slightly lower compared with the control group (97.3 % and 100 %, respectively).
These mean group values were disproportionately influenced by one female from the 120 mg/kg/day group, which delivered 10 stillborn pups out of 12 newborn pups and subsequently lost the 2 live pups. In the absence of similar case in the higher dose groups, this isolated finding in the low dose group was considered incidental.
The survival index was 100% in all groups (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
There was no effect of maternal treatment on mean pup body weight in any group for either sex (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
There was no test item-related effect on mean T4 concentration in PND13 pups (male and female) in any group (for more information, please refer to section "Any other information on results inlc. tables").
The mean normalized anogenital distance was slightly but statistically significantly longer for the males in the 350 and 1000 mg/kg/day groups compared with the concurrent control group. However, all values fell within the historical control range, so the differences were considered to be of no toxicological significance (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
No nipples were observed for the male offspring examined on PND13 in any group (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
No test item-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of Wistar Han rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment.
The mean number of implantation and percentage pre-birth (post-implantation) loss in treated groups were comparable with the control group.
There was no effect of maternal treatment on sex ratio in any group (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).
Developmental neurotoxicity (F1)
Developmental immunotoxicity (F1)
Details on results (F1)
Effect levels (F1)
#1
- other: No adverse effects observed
Target system / organ toxicity (F1)
#1
Results: F2 generation
General toxicity (F2)
Developmental neurotoxicity (F2)
Developmental immunotoxicity (F2)
Details on results (F2)
Effect levels (F2)
Target system / organ toxicity (F2)
Overall reproductive toxicity
#1
Additional information about applicability domain and reliability of (Q)SAR predictions
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Table 1. Mean Thyroid Hormone Concentrations for F0 males on the Day of Necropsy and F1 male and female Pups on PND13-15
| Group Number | Dose Level (mg/kg/day) | Values | F0 Males on the Day of Necropsy | F1 Male Pups on PND13-15 | F1 Female Pups on PND13-15 |
| T4 (ng/mL) | T4 (ng/mL) | T4 (ng/mL) | |||
| 1 | 0 | MEAN | 46.0 | 53.1 | 51.8 |
| SD | 6.3 | 6.1 | 5.1 | ||
| N | 10 | 10 | 10 | ||
| 2 | 120 | MEAN | 45.5 | 52.1 | 52.4 |
| SD | 4.6 | 7.2 | 6.1 | ||
| N | 10 | 8 | 8 | ||
| 3 | 350 | MEAN | 47.9 | 51.6 | 49.2 |
| SD | 7.0 | 6.1 | 3.7 | ||
| N | 10 | 10 | 10 | ||
| 4 | 1000 | MEAN | 45.3 | 51.4 | 50.0 |
| SD | 6.0 | 3.3 | 3.4 | ||
| N | 10 | 10 | 10 |
Overall remarks, attachments
Attachments
Applicant's summary and conclusion
No adverse effects were observed in a reproduction/developmental toxicity screening test (OECD 421, GLP) conducted in Wistar Han IGS rats. The NOAEL is at least 1000 mg/kg bw/day for systemic, reproductive, and developmental toxicity.
In a reproduction/developmental toxicity screening test (OECD 421, GLP), 3-methyl-1,3-butanediol (purity 99.9 %) was administered to 10 Wistar Han IGS rats/ sex/dose by daily gavage at dose levels of 0, 120, 350, 1000 mg/kg bw/day. The test item was administered to males from 14 days before mating, throughout mating and up to the day before necropsy (i.e., 35 days). It was administered to females from 14 days before mating, throughout mating, gestation and lactation and up to the day before necropsy (i.e., lactation day (LD) 13-15).
There were no test item related effects on mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance for the F0 Generation. No effects were observed during paternal and maternal gross necropsy investigations, on organ weights and microscopic observations.
No treatment related effects were observed on pups viability and litter sizes, weight and sex of pups, and physical developmental landmarks of pups (anogenital distance and nipple retention in males). There was no test item-related change in T4 concentration in adult males and in post natal day (PND) 13 pups of either sex in any group.
The NOAEL is at least 1000 mg/kg bw/day for systemic, reproductive, and developmental toxicity.
This study is acceptable and satisfies the guideline requirement for an OECD 421.