Reaction mass of propylidynetrimethanol and 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The results from a 90-day repeated dose study performed with Polyol TD did not indicate any adverse effects on reproductive organs, estrous cycles and sperm parameters.

Furthermore, in a prenatal developmental toxicity study with Polyol TD in rats, no adverse effects on sexual function and fertiliy were observed.

While there was a decrease in live offspring observed in the high dose group during another prenatal developmental toxicity study in rabbits as a second species, these effects occured in the presence of adverse effects in dams, including mortality, and were therefore considered to be secondary to maternal toxicity.

 

In addition, an extended one-generation reproductive toxicity study (OECD TG 443) according to Annex X, Section 8.7.3. is planned based on ECHA's decision and will be provided when data available. 

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

other justification

Justification for data waiving:

other:

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Results on reproductive organs, estrous cycles and sperm parameters were derived from a GLP-compliant sub-chronic toxicity study in rats, which was performed according to OECD TG 408.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant subchronic toxicity study (De Marzi, 2020), performed according to OECD TG 408, Polyol TD (in water) was administered to 10 Sprague Dawley rats/sex/dose once daily, 7 days per week for 13 consecutive weeks by oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. In addition, 5 rats/sex from the control and high dose group (1000 mg/kg bw/day) were included in a treatment-free recovery group for a period of 4 weeks post-dosing.

The following investigations relevant for effects on reproductive organs and fertility were performed: oestrous cycle, sperm analysis, organ weights, macroscopic observations and histopathological examinations of reproductive organs. There were no treatment-related anomalies in the oestrous cycle of treated females at the end of the treatment and recovery periods.

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated high dose females. No morphological changes were detected when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina. No differences were seen in the sperm analysis between males from the control and high dose groups, considering motility, morphology and concentration expressed as million sperm/gram caudal epididymal tissue. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. The qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle (Stages I- XIV) in all control and treated males of the high dose group. Therefore, the results from this 90-day repeated dose study did not indicate any adverse effects on reproductive organs, estrous cycles and sperm parameters that could potentialy influence reproductive function and fertility and it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg bw/day.

 

In addition, no evidence of any effects on reproductive function and fertility were observed in a prenatal developmental toxicity study in the rat performed according to OECD TG 414 (Sisti, 2020).

In this study, 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.

There were no effects observed that could potentially affect the pregnancy outcome. No relevant treatment-related change were observed at microscopic and macroscopic observations and for thyroid weight.

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.

 

In another prenatal developmental toxicity study in a second species, which was performed according to OECD TG 414 (Sisti, 2021), effects of Polyol TD were investigated in New Zealand White rabbits during pregnancy and embryo-foetal development. While there were some effects on pregnancy outcome observed in the high dose group, these effects occured in the presence of adverse effects in dams, including mortality, and were therefore considered to be secondary to maternal toxicity:

Polyol TD (in water) was administered to 25 female New Zealand White rabbits/dose by oral gavage at dose levels of 0, 50, 140 and 400 mg/kg bw/day from days 5 through 28 of gestation.

Signs of maternal toxicity were observed at 400 mg/kg bw/day as indicated by the reduction in body weight/body weight gain and food consumption which occurred during the gestation phase and confirmed by a decrease in absolute body weight gain. Two females aborted and three were found dead. Females receiving 50 and 140 mg/kg bw/day showed marginal signs of maternal toxicity with an evidence of decrease in body weight and absolute weight gain. However at these dose levels the treatment-related effects were transient occurrences and were not considered adverse. Based on these findings, a maternal NOAEL of 140 mg/kg bw/day was identified in this study. The overall pregnancy rates were similar in all groups and ranged between 88% in control and 92%, 100% and 96% in treated groups, respectively. Gravid uterus weight was statistically significant decreased in females at 400 mg/kg bw/day. The total of live foetuses maternally exposed at 400 mg/kg bw/day was decreased slightly, when compared to the control group, recording also a statistically significant decrease in litter weight, but not in mean foetal weight. The observed decrease in litter size and litter weight in the high dose group could be considered as a secondary response to maternal stress and toxicity. Based on these findings, a NOAEL of 400 mg/kg bw/day for developmental toxicity was identified in this study.

 

Effects on developmental toxicity

Description of key information

The effects of Polyol TD during pregnancy and embryo-foetal development were investigated in the rat after oral administration in a reliable GLP study conducted according to OECD 414. There were no adverse effects observed in the maternal animals and the effects observed in the foetuses were comparable to historical control data. 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, this being the highest dose tested.

 

In addition, the effects of Polyol TD were investigated in New Zealand White rabbits as a second species, after oral administration during pregnancy and embryo-foetal development in a reliable GLP study conducted according to OECD 414. A NOAEL (No Observed Adverse Effect Level) for maternal toxicity of 140 mg/kg bw/day was identified in this study. Two females aborted and three were found dead at the top dose. While there was a decrease in live offspring observed in the high dose group, these effects occured in the presence of the described adverse effects in dams, including mortality, and were therefore considered to be secondary to maternal toxicity. A NOAEL of 400 mg/kg bw/day for developmental toxicity was therefore identified in this study.

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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

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.

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

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

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

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

While effects were observed these effects were not considered adverse.

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

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

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

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

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-07-10 to 2021-06-18
In-life period: 2020-07-22 to 2020-09-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Chemical name: Reaction mass of 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol and propylidynetrimethanol
EC no. 904-153-2

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF

Details on test animals or test system and environmental conditions:

A total of 120 sexually mature virgin female New Zealand White rabbits SPF, 14 weeks of age, at least 2.5 kg body weight, was ordered from Charles River Laboratories Italia S.r.l. The males used were of the same strain from the same supplier and were at least 25weeks of age and more than 3.5 kg in bodyweight. An acclimatisation period of 70 days was then allowed before the start of treatment.

The rabbits were housed in a limited access facility. Animal room controls were set to maintain temperature and relative humidity at 19°C±2°Cand 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

At arrival, the animals were hosed up to 2 per cage (where possible) in polycarbonate/stainless steel cages with perforated NorylTM floor suspended over trays. After mating, females were individually housed in the same type of cage as described above. Each cage tray held absorbent paper which was inspected and changed as necessary.

Drinking water and food was supplied ad libitum.

A commercially available laboratory rabbit diet (2RB15,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study. There was
no information available to indicate that any non-nutrient substance likely to influence the
effect of the test item was present in the drinking water or the diet. No antibiotic or other
chemotherapeutic agents are present in the diet.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened water (by reverse osmosis)

Details on exposure:

All females were administered by oral gavage during the gestation period from Day 5 through Day 28 post coitum at 50, 140 and 400 mg/kg/day. The dose volume was set at 5 mL/kg body weight.

The required amount of substance was dissolved in the vehicle. The preparations were made daily (concentrations of 10, 28 and 80mg/mL) or for several consecutive days (up to 7 consecutive days), according to stability data obtained from analytical method validation study. The preparations were maintained under magnetic stirring for at least 30 minutes before and throughout the dosing procedure. Concentrations were calculated and expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The substance's analytical method was validated in the range from 1 to 200 mg/mL. The proposed preparation procedure for the test item was checked in the range from 1 to 200 mg/mL by chemical analysis (concentration) confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in SOPs for concentration (90-110%). In the same study and in the same range of concentration, the the stability of the preparations was verified and the final results were as follows:
– up to 28 hours at room temperature;
– after 8 days at + 2-8°C.

Samples of the preparations prepared during the first and last week of treatment of the current study were analysed to check the concentration. Chemical analysis was carried out. Results of the analyses were within the acceptability limits for concentration of solutions (90-110%). The validated software used for this activity was Empower® 2 Build No. 2154.

Details on mating procedure:

Females were introduced to non-sibling fertile males obtained from the same supplier. Each female remained with the male for at least 1 hour after at least 2 successful matings were observed, where possible. The day successful mating (visual inspection of the mating occurrence) was detected was considered Day 0 post coitum (or gestation Day 0).

Duration of treatment / exposure:

All females were administered by oral gavage during the gestation period from Day 5 through Day 28 post coitum.

Frequency of treatment:

All females were administered once daily by oral gavage during the gestation period from Day 5 through Day 28 post coitum.

Duration of test:

Day 5 through Day 28 post coitum

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle only - Water

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

140 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 mated female rabbits per does group.

Control animals:

yes, concurrent vehicle

Details on study design:

-Animal species selection rationale:
The New Zealand White rabbit was the species and strain of choice because it is accepted
by many regulatory authorities for reproductive toxicology studies and there are ample
experience and background data on this species and strain.

- Dose selection rationale:
Dose levels were selected based on information from a previous, non GLP-compliant study
in pregnant NZW rabbits in which the dose levels of 200, 400 and 800mg/kg/day were used.
At the dose level of 800mg/kg/day 4 females were humanly sacrifice for abortion, arrest of
pregnancy for a probable process of resorption of foetuses or for severe clinical signs observed.
Severe maternal toxicity was expressed by a decrease in the body weight and lower absolute weight
gain. The two females sacrificed at term showed a decrease in the average of gravid uterus
weight or in the mean foetal weight. At the dose level of 400mg/kg/day no clinical signs
were observed throughout the study, but limited maternal toxicity was noted during the
second part of the gestation phase, in terms of a decrease in body weight gain and absolute
weight gain. Mean foetal weight was also decreased. At the dose level of 200mg/kg/day, no
clinical signs and no changes in body weight were evident, but the absolute weight gain
resulted lower than the control group.

Maternal examinations:

Mortality:Throughout the study, all animals were checked early in the morning 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. This allowed post mortem examinations to be carried out during the working period of that day. A complete necropsy was performed.

Abortions: Animals that aborted were killed by an intravenous injection of a suitable euthanasia agent (Pentobarbital overdose or Tanax®. Tanax was injected in sedated animals) on the same day that the abortion was detected. A complete necropsy was performed in all cases.

Clinical signs: All clinical signs were recorded for individual animals. At least once daily during treatment, each animal was observed and any clinical signs recorded with the date and time of onset, duration and progress of the observed condition, as appropriate. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Body weight: Each group animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on Days 3, 5, 8, 11, 14, 17, 20, 23, 26 and 29 post coitum.

Food consumption: Food consumption was recorded on Days 3, 5, 8, 11, 14, 17, 20, 23, 26 and 29 of post coitum starting from Day 0 post coitum.

Necropsy: All animals, including those killed for humane reasons and those found dead, were subjected to necropsy and the number of implantations and corpora lutea were recorded if possible. The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination: The thyroid was fixed and preserved in 10% neutral buffered formalin. After dehydration and embedding in paraffin wax, section(s) of the thyroid were cut at 5 micrometre thickness and stained with haematoxylin and eosin. The examination was as detailed below:
– All animals in the control and high dose groups.
– All animals in the low and medium dose groups killed or dying during the treatment period.


Organ weight: From all females completing the scheduled test period, the thyroid was dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Ovaries and uterine content:

The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals found dead or killed during the study);
– Corrected maternal body weight (terminal body weight minus gravid uterus weight)
– Corrected maternal body weight gain (corrected maternal body weight minus maternal body weight on gestation Day 3)
– number of corpora lutea for pregnant animals;
– number of implantations for pregnant animals;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

All viable foetuses were euthanised, weighed and examined externally and internally. The thoracic and abdominal cavities were opened and examined and sex was determined. Head section (medial suture bones) was performed at necropsy on each foetus, selected for skeletal examination, and particular attention was paid to the brain ventricles. The head from approximately half of the foetuses (i.e. routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed examination of internal structures of the head: eye, brain, nasal passages and tongue. Foetuses were eviscerated, skinned and fixed in 95% ethanol for subsequent skeletal staining and examination. Skeletal and fixed head examinations were performed in all groups.

Structural deviations 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.

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 theWilliams test. The criterion for statistical significance was p<0.05.

Indices:

Pre impl. Loss %= (no. of corpora lutea−no. of implantations)/no. of corpora lutea*100%
Post impl. Loss%=(no. of implantations−no. of live foetuses)/no. of implantations*100%

Historical control data:

Historical control Litter data, Macroscopic observation of females at final cesarean section, female fate data, and foetal pathology data were gathered at the same test facility from 5 studies between 2014 and 2018.

Please see attachment 1 for details.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The main clinical sign noted in females sacrificed at termination, both control and treated, was reduced faeces. An increase incidence in females affected by this finding was evident at 400 mg/kg bw/day.
Signs of hairloss in different areas of the body surface, were seen among the treated groups without dose relationship.
Reduced water consumption were noted in one control female and two female from 400 mg/kg/day group.
Soft faeces on cage tray were noted on occasion in one female receiving 140 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three animals were found dead and another two animals were sacrificed for ethical reasons due to foetuses on cage tray/abortion at the top dose of 400 mg/kg bw/day.

One animal was sacrificed for ethical reasons due to foetuses on cage tray/abortion at the dose of 50 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average of the body weight did not differ between groups throughout the study, with the exception of a decrease seen on Day 29 post coitum in females receiving 400 mg/kg bw/day.

Treatment-related decrease in the body weight gain with or without significance at statistically analysis was evident at 400 mg/kg bw/day, when compared to the control group starting from Day 5 post coitum until termination, excluding Day 20. A transient decrement in body weight gain was also noted at 50 and 140 mg/kg bw/day on Days 17, 20, 23 followed by a partial trend of recovery.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Between Days 11 and 20 post coitum, food consumption was decreased in females receiving 400 mg/kg bw/day, sometimes with significance, at statistically analysis and up to -29%, compared to the control group.
Food consumption in the other two dose levels remained quite comparable to the control group during the gestation period.

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:

no effects observed

Description (incidence and severity):

There was no effect on the thyroid gland absolute and relative weights in treated females when compared to controls.

In the microscopic observation of the thyroid gland of the high does group animals and those females found dead or humane killed in the low and mid-dose group, no treatment-related changes were noted. The microscopic observations (ultimobranchial cyst/s) noted in control and treated animals were consistent known to occur spontaneously in untreated New Zealand White rabbits of the same age and were considered incidental and unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on the thyroid gland absolute and relative weights, in treated females when compared to controls.

Gravid uterus weight was statistically significant decreased in females receiving 400 mg/kg bw/day, compared to the control group. Dose-related decrease in absolute weight gain was noted in all treated group compared to the control. Corrected body weight was unaffected by treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the final sacrifice, no treatment related changes were noted of all treated females when compared to controls.

In unscheduled death animals (1animal in group 2 and 5 animals in group 4):
-No abnormalities were found in the Group 2 female and consequently the cause of death was not established.
-4 of Group 4 females had dark red staining in the urogenital region (3 out of 5) and/or multiple dark areas in the stomach (2 out of 5), distended gall bladder (2 out of 5) and in a single instance pale and red areas in the liver.
-The last Group 4 female, did not show any macroscopic changes anyway showed very poor health condition in the in vivo phase, which had determined the humanitarian sacrifice.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

In the microscopic observation of the thyroid gland of the high does group animals and those females found dead or humane killed in the low and mid-dose group, no treatment-related changes were noted. The microscopic observations (ultimobranchial cyst/s) noted in control and treated animals were consistent known to occur spontaneously in untreated New Zealand White rabbits of the same age and were considered incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One animal in the low dose (50 mg/kg bw/day) group and 2 animals in the high dose (400 mg/kg bw/day) group had foetuses on the cage tray.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The number of corpora lutea and implantation sites was similar between groups, although
a slight decrease, without significance at statistically analysis, was noted at 400 mg/kg/day.
Pre-implantation loss (expressed as percentage) was increased in females receiving 400 mg/kg bw/day, without significance at statistically analysis, when compared to the control. Also females receiving 50 and 140 mg/kg bw/day showed a similar trend , but these were contributed by two females (1 female in each does group) who showed a high value of pre-implantation loss (37.5% or 50%, respectively). However, this differences between the number of corpora lutea and number of implantations sites were considered unrelated to treatment, since animals were not treated during the implantation period (Days 0-5 of gestation).

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Intra-uterine deaths (early, late and total resorption) did not differ between control and
treated groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

One dead foetus was noted in one control female while none occurred at treated groups.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The overall pregnancy rates were similar in all groups and ranged between 88% in control and 92%, 100% and 96% in treated groups, respectively.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

140 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

number of abortions

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Mortality/abortion

Description (incidence and severity):

Three animals were found dead and another two animals were sacrificed for ethical reasons due to foetuses on cage tray/abortion at the top dose of 400 mg/kg bw/day.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant change in mean feotal weight (for each sexes, as well as for both sexes combined).

An increase incidence in small foetuses (body weight < 35g) were noted at the dose levels of 140 and 400 mg/kg bw/day with lack of dose relationship .

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of live foetuses maternally exposed at 400 mg/kg bw/day was slight decreased, when compared to the control group.

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 44% and 48%.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in litter weight was observed at 400 mg/kg bw/day. The observed slight decrease in litter size and litter weight in the high dose group could be considered as a secondary response to maternal treatment-related effects with the test item expressed as abortion, reductions in maternal weight gain and absolute weight gain. In addition the decrease in litter size was without correlation with the dose level.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The two major abnormalities noted in one foetus maternally exposed at 50 mg/kg bw/day that showed absence of tail and in three foetuses maternally exposed at 140 mg/kg bw/day that showed hyperextension of the hindlimbs were considered unrelated to treatment in the absence of a dose relationship.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No malformations were recorded among the treated groups.

The minor alterations, classified as variations or anomalies, were found both in control and treated groups without substantial differences.

A slight increase in incomplete ossification of the sternebrae, metatarsal/distal or proximal phalanges of the hindlimbs bones were noted in treated foetuses compared to the control. In addition some of these were also found in small foetuses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Common alterations (dilated lateral cerebral ventricles or folded retina) were equally distributed between control and treated groups. Blackish mass in the brain was seen in treated foetuses/litters. This observation, which was also observed, with a high incidence in the head of control foetuses could be considered to represent an artificial finding and then not treatment-related.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Remarks on result:

other: No teratogenicity toxicity found at skeletal and visceral examination. Decrease in litter size and weight considered secondary to maternal effects.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Maternal data in tabular form are presented in Attachment 2.

Litter data in tabular form are presented in Attachment 3.

Fetal data in tabular form are presented in Attachment 4.

Conclusions:

A NOAEL (No Observed Adverse Effect Level) for maternal toxicity of 140 mg/kg bw/day was identified in this study based on reduction in body weight/body weight gain and food consumption which occurred during the gestation phase and confirmed by a decrease in absolute body weight gain. Two females aborted and three were found dead at this dose.

A NOAEL of 400 mg/kg bw/day for developmental toxicity was identified in this study - this being the highest tested dose. No development toxicity in terms of teratogenicity was noted since no relevant alterations were found at skeletal and visceral head examinations. The slight decrease in litter size and litter weight observed at this dose level were considered to be a result of severe maternal toxicity.

Executive summary:

In a developmental toxicity study performed according to OECD TG 414, the substance (in water) was administered to 25 female New Zealand White rabbits/dose by oral gavage at dose levels of 0, 50, 140 and 400 mg/kg bw/day from days 5 through 28 of gestation.

Signs of maternal toxicity were observed at 400 mg/kg bw/day as indicated by the reduction in body weight/body weight gain and food consumption which occurred during the gestation phase and confirmed by a decrease in absolute body weight gain. Two females aborted and three were found dead. Females receiving 50 and 140 mg/kg bw/day showed marginal signs of maternal toxicity with an evidence of decrease in body weight and absolute weight gain. However at these dose levels the treatment-related effects were transient occurrences and were not considered adverse. No treatment related findings were seen at necropsy including the thyroid weight. In addition, the histopathology of thyroid did not reveal pathological changes. Based on these findings, a maternal maternal NOAEL of 140 mg/kg bw/day was identified in this study.

The overall pregnancy rates were similar in all groups and ranged between 88% in control and 92%, 100% and 96% in treated groups, respectively. Gravid uterus weight was statistically significant decreased in females at 400 mg/kg bw/day. Pre-implantation loss was increased in females receiving 400 mg/kg bw/day, but considered as treatment related. The total of live foetuses maternally exposed at 400 mg/kg bw/day was decreased slightly when compared to the control group, recording also a statistically significant decrease in litter weight, but not in mean foetal weight. The observed decrease in litter size and litter weight in the high dose group could be considered as a secondary response to maternal stress and toxicity. No evidence of teratogenicity was seen at any dose level, in terms of developmental toxicity in foetuses recorded at fixed visceral (head) and skeletal examinations. Based on these findings, a NOAEL of 400 mg/kg bw/day for developmental toxicity was identified in this study.

 

This developmental toxicity study in the rabbit is considered acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbits.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

140 mg/kg bw/day

Species:

rabbit

Quality of whole database:

Results were derived from two GLP-compliant prenatal developmental toxicity study in a first (rat) and second (rabbit) species, which were performed according to the relevant OECD TG 414.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

In summary, there were no adverse effects observed on offspring development in the first species (rats) up to the highest dose tested (1000 mg/kg bw/day).

 

In a developmental toxicity study performed according to OECD TG 414, Polyol TD (in water) was administered to 25 female New Zealand White rabbits/dose by oral gavage at dose levels of 0, 50, 140 and 400 mg/kg bw/day from days 5 through 28 of gestation.

Signs of maternal toxicity were observed at 400 mg/kg bw/day as indicated by the reduction in body weight/body weight gain and food consumption which occurred during the gestation phase and confirmed by a decrease in absolute body weight gain. Two females aborted and three were found dead. Females receiving 50 and 140 mg/kg bw/day showed marginal signs of maternal toxicity with an evidence of decrease in body weight and absolute weight gain. However at these dose levels the treatment-related effects were transient occurrences and were not considered adverse. No treatment related findings were seen at necropsy including the thyroid weight. In addition, the histopathology of thyroid did not reveal pathological changes. Based on these findings, a maternal NOAEL of 140 mg/kg bw/day was identified in this study.

The overall pregnancy rates were similar in all groups and ranged between 88% in control and 92%, 100% and 96% in treated groups, respectively. Gravid uterus weight was statistically significant decreased in females at 400 mg/kg bw/day. Pre-implantation loss was increased in females receiving 400 mg/kg bw/day. The total of live foetuses maternally exposed at 400 mg/kg bw/day was decreased slightly, when compared to the control group, recording also a statistically significant decrease in litter weight, but not in mean foetal weight. The observed decrease in litter size and litter weight in the high dose group could be considered as a secondary response to maternal stress and toxicity. No evidence of teratogenicity was seen at any dose level, in terms of developmental toxicity in foetuses recorded at fixed visceral (head) and skeletal examinations. Based on these findings, a NOAEL of 400 mg/kg bw/day for developmental toxicity was identified in this study - this being the highest tested dose.

In summary, the observed decrease in litter size and litter weight in the high dose group could be considered as a secondary response to maternal stress as it occured in the presence of maternal toxicity and mortality. These effects are therefore not considered relevant for classification for reproductive toxicity of the test compound.

Justification for classification or non-classification

Based on the findings from reliable prenatal developmental toxicity studies in two species (rat and rabbit) which were conducted with the test substance itself, no classification for developmental toxicity is proposed according to Regulation (EC) No. 1272/2008. Some effects on litter size and litter weight were seen in the high dose group of the rabbit developmental study but are considered as a secondary response to maternal stress as they occurred in the presence of maternal toxicity and mortality. These effects are therefore not considered relevant for classification for developmental toxicity of the test compound. No evidence of developmental toxicity was seen at any dose level.