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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-01-13 to 2020-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
yes
Remarks:
At arrival, the weight range for males was 331 to 335g instead of at least 340g as per the Study Protocol. This deviation was not considered to have affected the integrity or the purpose of the study. No other deviations occurred during the study.
GLP compliance:
yes
Limit test:
no

Test material

1
Reference substance name:
(5-ethyl-1,3-dioxan-5-yl)methanol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethylpropane-1,3-diol
EC Number:
904-153-2
IUPAC Name:
(5-ethyl-1,3-dioxan-5-yl)methanol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethylpropane-1,3-diol
Details on test material:
Polyol TD, batch no. 7350 was described as a clear liquid, and stored in sealed container under dryed gas (Nitrogen) blanket at ambient temperature in dry area protected from direct sunlight and the elements. The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor.
Specific details on test material used for the study:
Identity: Polyol TD
Chemical name: Reaction mass of 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol and propylidynetrimethanol
Appearance: Clear, colourless liquid
Batch no: 7350
Inspection No: 890000050160
EC no: 904-153-2
Expiry date: 18 June 2020
Storage conditions: Store in sealed container under dry gas (Nitrogen, Argon or dried air) blanket, at ambient temperature, in dry area protected from direct sunlight and the elements

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age and weight at study initiation: females - 9 weeks old (200-225 g) and males at least 11 weeks old (at least of 331 g):
- Housing: The animals were housed in a limited access rodent facility. During the pairing period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): rooms were lit by artificial light for 12 hours each day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparations of the test item were prepared as solutions in purified water. Concentration was assessed for all levels by taking two analytical aliquots (approximately 1 mL). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the two determinations.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed once a day from Day 3 through Day 19 post coitum.
Frequency of treatment:
All animals were dosed once a day from Day 3 through Day 19 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle - water
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:
25 mated females/group
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Mortality: Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical Signs: All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.

Body weight: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption: Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

Clinical pathology investigations: Blood collection was performed, for hormone determination, from all females at termination on Day 20 post coitum. Blood samples (approximately 0.5 mL) were withdrawn from the sublingual vein under light isoflurane anaesthesia. This procedure was performed within a short timeframe (e.g. two hours, as possible) on the morning of the day of necropsy. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH).

All animals were subjected to necropsy, supervised by a pathologist. From all females, the organs that were found to be abnormal and the thyroid were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. These organs were subject to histpathological examination.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight, including the cervix;
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– internal foetal sex determination in each foetus allocated to the skeletal examination; internal foetal sex determination for the remaining foetuses performed during the fixed-visceral examination;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– anogenital distance (AGD) in all live foetuses;
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– Early resorptions: only placental remnants visible.
– Late resorptions: placental and foetal remnants visible.
Blood sampling:
Clinical pathology investigations: Blood collection was performed, for hormone determination, from all females at termination on Day 20 post coitum. Blood samples (approximately 0.5 mL) were withdrawn from the sublingual vein under light isoflurane anaesthesia. This procedure was performed within a short timeframe (e.g. two hours, as possible) on the morning of the day of necropsy. Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH).
Fetal examinations:
All foetuseswere examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed visceral examination.

Sex determination performed during post mortem examination was confirmed by internal gonads inspection, during the fixed-visceral examination. Internal foetal sex determination in each foetus allocated to the skeletal examination was performed during the post mortem examination. Indication of incomplete testicular descent/cryptorchidism was noted in male foetuses.

The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination (single staining). Skeletal and fixed-visceral examinations were performed in all groups. Structural deviation were classified as follows:
Malformations: Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies: Minor abnormalities that are detected relatively frequently.
Variants: A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Pre-implantation loss

Anogenital distance
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Historical control data:
Historical control data was used for thyroid hormone determination, for comparison with foetal examinations, sex ratio and litter data and for pregnancy information.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse clinical signs were observed throughout the study both in control and treated females.

Four control and one receiving 300 mg/kg bw/day showed scab on neck or head. In addition, two control females and two receiving 300 mg/kg/day showed hairloss on head. These signs were in general observed during the middle or last gestation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animals died during the study.

One female receiving 1000 mg/kg bw/day was found not pregnant at necropsy.
One control female had unilateral implantation in the left horn and was not pregnant in the right one.
The number of females with live foetuses on Day 20 post coitum was: 25 in the control, 25 in the low (100 mg/kg bw/day - Group 2), 25 in the medium (300 mg/kg bw/day - Group 3) and 24 in the high dose groups (1000 mg/kg bw/day - Group 4).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No relevant differences in body weight were noted between control and treated groups. A very slight statistically significant decrease in mean body weight (-4%) was noted on Day 6 post coitum, in females receiving 300 and 1000 mg/kg bw/day, when compared to the control mean values. On Day 6 post coitum, body weight gain was decreased (-8%, -22%, -38% in order of ascending dose levels) at all dose levels, reaching a statistically significance at 1000 mg/kg bw/day, compared to the control group. This change was considered related to treatment but not adverse since occurred as isolated case and afterwards, a regular increment was noted in all treated groups compared to the control.

No significant differences in terminal body weight were observed in treated groups compared to the control group. A slight decrease (-9%) in absolute weight gain (terminal body weight minus body weight at Day 0 post coitum minus gravid uterus), was noted in females receiving 1000 mg/kg bw/day, when compared to control group. Due to the limited entity of this change it was considered to be without toxicological relevance.

Please see results table below.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A reduction (-16%) in food consumption, statistically significant on Day 6 post coitum, was noted in females receiving 1000 mg/kg bw/day, compared to the control group. This was considered treatment-related, but not adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
No dose-related changes were recorded.

Compared with controls, females dosed at 1000 mg/kg bw/day showed a statistically significant increase of thyroxine (16%). Looking at individual data, only four animals showed thyroxine data outside the historical range and simultaneous change of the other parameters (decrease of TSH and increase of T3) was recorded in one animal only. Since the simultaneous changes of the three hormones were sporadic, and no histopathological changes were recorded, the above findings were considered to be incidental.

T3 and TSH values were outside the range of historical data in some control and treated animals, therefore these findings were considered to be unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on the thyroid weight (absolute and relative) was noted at any dose levels, compared to the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Females that completed the treatment period did not show relevant macroscopic changes that could be considered treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.

The sporadic lesions, reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Please see data presented below.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One female receiving 1000 mg/kg bw/day was found not pregnant at necropsy.

Please see details below.
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
While effects were observed these effects were not considered adverse.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter weight and mean foetal weight (for each sexes, as well as for both sexes combined) of treatment groups were comparable to the control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratios of the foetuses, calculated as the percentage of males per litter, did not show differences between control and treated groups and ranged between 49% and 53%.

Please refer to table below for details.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter weight and mean foetal weight (for each sexes, as well as for both sexes combined) of treatment groups were comparable to the control group.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No relevant differences were noted in the mean values of the AGD (anogenital distance normalised for the cube root of the body weight performed on Day 20 post coitum) of foetuses of both sexes maternally exposed at all dose levels compared to the control group.

A statistically significant increase was noted in males at the dose level of 1000 mg/kg bw/day compared to the control group. However, the difference was very slight, +3% compared to controls.

No such effect was observed in females.

Please attached document for details.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One control foetus showed some alterations recorded at the external examination classified as malformation on the jaw (micromaxilla), or anomalies on the tail (bent), on the head (domed shape), on the hindlimb (swollen with flexure in the left one and hyperextension in the right one). In addition, one control foetus showed a subcutaneous hematoma on the right side of the head (anomaly). One foetus of dam maternally exposed at 1000 mg/kg bw/day had an imperforate anus (malformation) and rudimentary tail (anomaly).

A total of 5 small foetuses (< 2.7 g) were detected, 1 out of 377 in the control group, 1 out of 371 in the low dose group and 3 out of 372 in the medium group.

Please refer to the attachment on external examination of foetuses data.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Major alterations were distributed along the treated groups without dose relationship, in terms of foetuses/litters affected. Please see table below for details.

Regarding the other alterations (anomalies and variations), unossified or incomplete ossification of various part of the skeleton were noted in control and treated groups without differences. These findings involved the forepaw (no ossification of the 4th metacarpal), sternebrae elements (incomplete ossification or no ossification of the 5th and/or 6th), bones of the skull (incomplete ossification of the supraoccipital, interparietal and temporal segments, no ossification or incomplete ossification of the hyoid), sacral vertebrae (incomplete ossification of the arch(es)). The type and distribution of the above mentioned findings, both for major and minor alterations, did not show any association with treatment with the test item.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The major alterations (malformations) noted, at visceral examination of foetuses, were on the brain/head. These included the extreme enlargement of the lateral ventricles of the brain, at dose levels of 100 and 300 mg/kg bw/day and a cerebral hypoplasia associated to the absence of the third ventricle at the dose level of 1000 mg/kg bw/day. One foetus maternally exposed at 300 mg/kg bw/day showed cleft palate. Although an increase in malformation rate was noted in all treated groups, the alterations were quantitatively similar between doses, occurred in foetuses without growth retardation and with an absence of doses-related response when compared to the control group. However, the brain alteration is present in the background control data (see attached document), even if with a severity between slight to moderate. Please refer to table below for details.

One foetus maternally exposed at 100 mg/kg bw/day showed a small opened area in olfactory bulb (classified as anomaly). In addition, alterations recorded in the ureters (enlarged and kinked), testis (displaced) and kidneys (ectopic), classified as variations and anomalies, were observed in control and treated groups with similar incidence in terms of litter and foetuses affected.
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Implantation Loss Data (%):

 Group    Pre-implantation loss (%)  Post-implantation loss (%)  Total (%)
 1  Mean  3.61  2.06  5.66
   SD  10.73  3.91  10.72
   (n)  25  25  25
 2  Mean  3.21 0.98   4.14
   SD  5.89  2.88  6.56
   (n)  25  25  25
 3  Mean  1.89  4.00  5.84
   SD  3.56  9.92  10.10
   (n)  25  25  25
 4  Mean  5.03  2.51  7.38
   SD  9.03  6.38  11.05
   (n)  24  24  24

Fate of Females

   Group 1 (control)  Group 2  Group 3  Group 4
 Initial group size  25  25  25  25
 Unilateral implantation  1  0  0  0
 Not pregnant 0   0  0  1
 With live foetuses at gestation Day 20 25   25  25  24

Terminal Body Weight, Gravid Uterus Weight and Absolute Weight Gain Data

   Terminal body weight (g)  Gravid uterus weight (g)  Absolute weight gain (g)
 1  Mean  410.18  88.50  63.44
   SD  28.88  19.52  12.24
   (n)  25  25  25
 2  Mean  413.86  88.47  69.08
   SD  31.31  11.02  13.88
   (n)  25  25  25
 3  Mean  399.78  88.91  60.23
   SD  26.50  12.92  14.98
   (n)  25  25  25
 4  Mean  396.29  85.57  57.43
   SD  32.29  16.09  14.69
   (n)  24  25  24

Skeletal examination of foetuses

 Groups (mg/kg bw/day) 1 (0)   2 (100)  3 (300)  4 (1000)
 Malformations        
 Forelimb(s): Humerus misshapen  2 (2) 0   0  0
 Pectoral girdle: Clavicle malpositioned  1 (1) 0 0 0
 Pectoral girdle: Scapula misshaped  0  0  2(1)  0
 Pelvic girdle: Pubis no ossification  4 (3)  3 (3)  4 (4)  3 (2)
 Pelvic girdle: Ilium no ossification  0  0  1(1)  0
 Skull: Tympanic annulus malposition  1(1)  0  0  0
 Skull: Zygomatic malpositioned  2 (2)  0  1(1)  2(2)
 Thoracic vertebrae: Arch(es) fused  0  0  3(1)  0
 foetal incidence; ( ) litter incidence           

Visceral examination of foetuses Data

Group (mg/kg bw/day) 1 (0)   2 (100)  3 (300)  4 (1000)
 Malformations        
 Brain: Ventricles enlarged extreme  0 1(1)    1(1)  0
 Brain: Cerebral hypoplasia and third ventricle absent  0 0 0  1(1)
 Head: Cleft palate  0  0   1(1)  0
 foetal incidence; ( ) litter incidence           

Applicant's summary and conclusion

Conclusions:
Based on the findings from this study, the NOAEL (No Observed Adverse Effect Level) for both maternal and developmental toxicity could be set at 1000 mg/kg bw/day.
Executive summary:

In a developmental toxicity study performed according to OECD TG 414, Polyol TD (in water) was administered to 25 mated female Sprague Dawley rats/dose by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 3 through 19 of gestation.

No mortality in the maternal animals occurred during the study and animals did not show treatment-related clinical signs. Maternal body weight gain was reduced in treated groups on occasion (Day 6 post coitum). In addition food consumption was also decreased in females receiving 1000 mg/kg bw/day. These parameters tended to recover, were comparable between control and treated groups during the last gestation period and did not affect the pregnancy outcome. Therefore, these effects were considered treatment-related but not adverse. No relevant treatment-related change were observed at microscopic and macroscopic observations and for thyroid weight. The sporadic changes of the three hormones (T3, T4 and TSH) determined in the parental females were considered to be unrelated to treatment.

No relevant differences were noted in the mean values of the anogenital distance of foetuses of both sexes compared to the control group. No differences were observed between control and treated groups in the number of implantation sites, corpora lutea, implantation losses, uterine deaths, viable foetuses, mean foetal weight (for each sex as well as for both sexes combined) and sex ratios. One foetus maternally exposed at 1000 mg/kg bw/day showed, at external examination, an imperforate anus (malformation) and rudimentary tail (anomaly). At skeletal examinations, the type and distribution of the findings observed, both for major and minor alterations, did not show any association with treatment with the substance. Although an increase in malformation rate was noted, at visceral examination, in all treated groups, the alterations were quantitatively similar between doses, occurred in foetuses without growth retardation and with an absence of doses-related response when compared to the control group.

Based on the findings from this study, the NOAEL (No Observed Adverse Effect Level) for both maternal and developmental toxicity is set at 1000 mg/kg bw/day, this being hte highest dose tested.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.