2-amino-2-ethylpropanediol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined repeated dose and reproduction/developmental toxicity screening study performed according to OECD 422 to assess the potential reproductive and fertility effects of 2-amino-2-ethyl-1,3-propanediol (AEPD) is available (Ishida, 2004). In this study doses of 250, 500 and 1000 mg/kg bw/day of the test substance were administered by gavage to rats during pre-mating, mating, gestation and until lactation day 4. A satellite group with a recovery period of 14 days after dosing ended was included in the control and 1000 mg/kg bw/day groups. As no effects were observed up to and including the highest dose level, the NOAEL for systemic toxicity is considered to be > 1000 mg/kg bw/day. In the P-generation, the parental reproduction parameters (including mating index, fertility index, gestation period, delivery index, and number of live pups) were not affected by treatment with the test substance up to and including the highest dose level of 1000 mg/kg bw/day. The NOAEL for reproduction is therefore considered to be > 1000 mg/kg bw/day.

There are no further animal data available on toxicity to reproduction of AEPD. The members of the category of 2-amino-1,3-propane-diols are substances that share a common propane backbone with an amine group at 2-carbon position and primary alcohols at 1 and 3 positions. The following substances are thus members of the aminopropanediol category: 2-amino-2-ethyl-1,3-propanediol (AEPD, CAS No. 115-70-8), 2-amino-2-methyl- 1,3-propane-diol (AMPD, CAS No. 115-69-5), 2-amino-1,3-propanediol (APD, CAS No. 534-03-2) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (trometamol, CAS No. 77-86-1) The only structural difference between trometamol and AEPD is a replacement of a hydroxyl group with a methyl group. Further analogues differ in the length of the alkyl side-chain at position 2 so that the following sequence is obtained: from 0 carbon atoms (APD) through 1 (AMPD) to 2 (AEPD). There are no other functional groups present in these molecules. 

 

The modelling of potential metabolites via the OECD QSAR toolbox v.2.0 (2010) did not predict relevant metabolites of the category members. Based on the chemical structure of the parental compounds, no metabolism is expected. Therefore, it can be assumed that aminopropanediols will not show reactive properties under in vitro and in vivo test conditions. All the category members are of low concern with regard to systemic toxicity. Available studies via the oral, dermal and intraperitoneal route indicate low acute and repeated dose toxicity. Inhalation is of no concern, because the low vapour pressure means that exposure is unlikely to occur. The results of the acute studies, as well as the repeated dose studies, demonstrate that the main cause of toxicity was the intrinsic alkalinity of the category members at the site of contact. The Cramer classification (related mainly to the oral route) also indicates a low toxicological concern for all the category members. No metabolism by cytochrome P450 enzymes in vivo is expected; this is supported by predictions from QSAR modelling. 

 

Data on selected reproduction parameters are available from the 90-day repeated dose toxicity study (oral gavage) with APD in rats, performed according to OECD 408. To assess the possible effect on reproduction, additional parameters were included (extensive gross necropsy and detailed histopathology of male and female reproductive organs and tissues, and sperm parameters). The available data indicate that no toxicity to reproduction is expected. Furthermore, no additional studies are justified for this endpoint based on animal welfare considerations.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

ovary weights were not recorded

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crj:CD9SD)IGS SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan Co., Japan
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: (P) 345–403 g (males) and 206–258 g (females)
- Fasting period before study: No
- Housing: The animals were housed individually in metal mesh cages. During the mating period, two rats, one male and one female, were kept in one cage. They were then kept individually in plastic Ekon cages with flooring (white flakes; Charles River Japan Co.) from day 17 of pregnancy to day 4 of lactation.
- Diet: NMF pellets (Oriental Yeast Co.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted with water for injection to make the administration volume 10 mL/kg bw, and 25, 50 and 100 mg/mL solutions were prepared. The preparations were performed in quantities for a maximum of 7 days, and the quantities for one day were put into brown glass bottles (light-proof bottles) and stored under refrigeration (measured temperatures 3–5 °C).

VEHICLE
- Concentration in vehicle: 25, 50 and 100 mg/mL

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Females were housed individually in plastic Ekon cages with flooring (white flakes; Charles River Japan Co.) from day 17 of pregnancy to day 4 of lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the solutions used in the administration were measured twice by GC, before the first administration and in the final week of the administration, and were found to have suitable concentrations.

Duration of treatment / exposure:

Males: 42 days
Females: 42-48 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

All of the females confirmed to have mated were allowed to deliver naturally and any difficulties delivering were noted. The completion of delivery was checked twice a day (a.m. and p.m.) up to from day 21 of pregnancy to the afternoon of day 22 of pregnancy and the pregnancy period was calculated in the unit of 0.5 days. If the deliveries were completed at 5:00 p.m., that day was taken as day 0 of nursing; the pregnancy period (days) (day 0 of nursing–day 0 of pregnancy) and the delivery rate (%) [(number of females delivering surviving pups)/(number of pregnant females) x 100] were calculated.
The incidence of muricide of the delivered pups and the treatment of the placentas and amnia were observed and the day of the delivery was noted as day 0 of lactation. The surviving pups were nursed up to lactation day 4 and the succes rate was observed, using the picking up of the pups, taking care of the nests, and the nursing as parameters.

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg
Basis:
other: nominal dose

No. of animals per sex per dose:

12 animals per sex per dose were used in the 0, 250, 500 and 1000 mg/kg bw groups. In addition, a satellite group of 5 animals per sex was included in the control and 1000 mg/kg bw groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a range-finding study during which rats were administered 125, 250, 500 and 1000 mg/kg bw for 2 weeks, no effects of the test substance were observed. 1000 mg/kg bw is the recommended maximum dose according to OECD test guideline 422. Based on this recommendation and the results of the range-finding study, 3 doses were selected, with 1000 mg/kg bw as the highest dose and 500 and 250 mg/kg bw as additional doses, using a geometric ratio of 2. Furthermore, satellite groups of 5 males and 5 females were established for the control and 1000 mg/kg bw groups.
- Post-exposure recovery period in satellite groups: 14 days

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during the dosing period and once daily during the recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
Cage side: signs of abnormal posture, convulsions, and abnormal behavior were observed.
During handling: ease of removal from the cage, reactions to handling, vocalisation during handling, state of fur and skin (dirty fur, rough fur, wounds, skin colour, etc.), eyes (protrusion of the eyeballs, eyelid closure), discharge from eyes and nose, mucous membranes, autonomous nerve functions (lacrimation, salivation, piloerection, pupil size, respiration) was noted.
In open field: gait, posture, lethargy, tremors, convulsions, rearing, urination, defecation, stereotypy (excessive grooming, circling, etc.), abnormal behavior (self-mutilation, walking backwards, etc.)
- Time schedule: Once before the administration started and once weekly thereafter, during the administration period and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the rats which were confirmed to have mated were measured twice a week (3 times in the first week of the administration and the first week of recovery) throughout the administration period (for all groups) and the recovery period (for satellite groups). For males, this was on administration day 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42. Males in the satellite group were also weighed on day 1, 4, 8, 11 and 14 of the recovery period. For females in the main groups, weighing was performed on administration day 1, 4, 8, 11 and 15 of pre-mating; day 1, 4, 7, 11, 14, 17 and 20 of pregnancy; and day 0 and 4 of lactation. Females in the satellite groups were weighed on the same days as were the males in the satellite groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumption was measured twice a week (3 times in the first week of administration) throughout the administration and recovery periods. For males, this was on administration day 1, 4, 8, 11, 15, 25, 29, 32, 36, 39 and 42. For females in the main groups, weighing was performed on administration day 1, 4, 8, 11 and 15 of pre-mating; day 1, 4, 7, 11, 14, 17 and 20 of pregnancy; and day 2 and 4 of lactation. Males and females in the satellite groups were weighed on administration day 1, 4, 8, 11, 15, 25, 29, 32, 36, 39 and 42; and day 1, 4, 8, 11 and 14 of the recovery period.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the sacrifice on the day after the final administration and the day after the end of the recovery period, laparotomies were performed on the animals under ether anesthesia and blood was taken from the abdominal aorta in sample bottles to which an anticoagulant (EDTA-2K) had been added.
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, overnight (approximately 16 hours)
- How many animals: 5 males and females from each group
- Parameters examined: red blood cell count, hemoglobin, hematocrit (calculated from the mean red blood cell volume and red blood cell count), mean red blood cell volume, mean red blood cell hemoglobin quantity (calculated from the hemoglobin quantity and the red blood cell count), mean red blood cell hemoglobin concentration (calculated from the hemoglobin quantity and the hematocrit value), platelet count, white blood cell count, reticulocyte percentage and white blood cell percentage.
Some blood was added to a container with 3.8% sodium citrate and centrifuged (3000 rpm, 10 minutes). The plasma was used to measure the prothrombin time, the activated partial thromboplastin time, and fibrinogen quantity.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the abdominal aorta at the same time as the samples for the hematologic tests; during the sacrifice on the day after the final administration and the day after the end of the recovery period.
- Animals fasted: Yes, overnight (approximately 16 hours)
- How many animals: 5 males and females from each group
- Parameters examined: after centrifuging the blood (3000 rpm, 10 minutes), the serum was used to measure the apolipaseprotein (AlP), total cholesterol, triglycerides, phospholipids, total bilirubin, glucose, urea nitrogen, creatinine, sodium, potassium, chlorine, calcium, inorganic phosphorus, total protein, albumin, and A/G ratio (calculated from the total protein and albumin values). AST, ALT, LDH and g-GTP were measured using the plasma from centrifuging heparinized blood (3000 rpm, 10 minutes).

URINALYSIS: Yes, males only
- Time schedule for collection of urine: The male animals were housed for 24 hours in metabolic cages during the last week of administration (day 38–39 of administration) and during the last week of recovery (days 9–10 of recovery).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for the first 4 hours the animals were fasted with access to water, during which urine was collected. For the remaining 20 hours, the rats had access to water and food ad libitum and the urine produced was collected separately from the 4-hour samples.
- Parameters examined: In the 4-hour samples; pH, protein, ketone bodies, glucose, occult blood, bilirubin, and urobilinogen (using Aution Sticks 7EA test paper, Arkray), color (by observation), and sediment (by microscopic observation). The osmolality of the 20-hour samples were measured (freezing point depression method, Auto & Stat OM-6030 automatic osmotic pressure measurement device, Arkray) and the 24-hour urine quantities were calculated from the 4-hour and 20-hour urine quantities. The 24-hour water consumption quantities were measured from the day before, using the water supply bottles in the metabolic cages.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last week of administration (males) and on day 4 of lactation after the sacrifice of the offspring (females) in the main groups. During the second week of recovery in the satellite groups.
- Dose groups that were examined: 5 males and 5 females from each group (main groups and satellite groups)
- Battery of functions tested:
Auditory response, approach response, touch response, pain response (tail pinch), pupillary reflex, aerial righting reflex and fot splay. Grip strength of the forelegs and hind legs was measured (spontaneous movement sensor for experimental animals Model NS-AS01, Neuroscience Co.). The measurements were performed for one hour; the number of movements were measured at 10-minute intervals and the total for 60 minutes were calculated.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily after the observation of the estrous cycle up to the day of group allotment and continuing until the confirmation of mating. The smears were examined microscopically. The vaginal smear images were classified into the pre-estrous, estrous, post-estrous, and estrous dormancy periods during the premating administration period; the number of days until the next estrous cycle was investigated on the basis of the number of appearances of the estrous cycle parameters and the number of estrous cylces was determined. Only animals with a normal estrous cycle were used in the study.

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
testis weight, epididymis weight.

Litter observations:

PARAMETERS EXAMINED
The numbers of surviving pups and stillborn pups were counted on the day of delivery. Abnormalities in the external appearances of the pups were observed, the numbers of each sex were counted, and their body weights were measured; after this, the dams were allowed to nurse them. The rate of still births (%) [(number of stillbirths/total number of pups delivered) x 100], the birth rates [(number of surviving pups/total number of pups delivered) x 100], external abnormality rates (%) [(number of pups with external abnormalities/number of surviving pups) x 100], and the sex ratios [number of males/(number of males+number of females)] were calculated. The stillbirths (except for the cases of marked post-mortem changes) were fixed in phosphate buffer 10 vol% formalin solution and stored. The surviving pups were observed once a day up to day 4 of nursing to see if they were still alive; the live-born survival rates (%) [(number of pups surviving on day 4 of nursing)/(number of live pups on day 0 of nursing) x 100] were calculated. Body weights of the live pups were measured on day 4 of nursing.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals in the main groups were sacrificed on day 42 of the study. All surviving animals of the satellite groups were sacrificed on day 14 of the recovery period.
- Maternal animals: All surviving animals in the main groups were sacrificed on lactation day 4. All surviving animals of the satellite groups were sacrificed on day 14 of the recovery period.

GROSS NECROPSY
- A detailed necropsy was performed, starting with a gross examination of all of the organs and tissues of the body, including the outer surface of the body, the head, the chest, and the abdomen. The numbers of corpora lutea and implantation sites of the female animals (dams) were counted. The absolute and relative weights of the organs listed below were measured for the 5 males and 5 females of each group from which hematologic and blood chemical test blood samples were taken (with the exception of the testes and epididymides, where all of the males were included). The bilateral organs indicated with asterisks were evaluated by measuring the weights of both the left and right organs and using the sum of these weights. Organs weighed: Brain, thyroids (including parathyroids), thymus, heart, liver, spleen, kidneys, adrenals, testes, and epididymides.

HISTOPATHOLOGY / ORGAN WEIGHTS
The whole organs and tissues listed below were taken from all the animals, fixed with phosphate buffer 10% formalin solution, and stored. Testes and epididymides were fixed with Bouin’s solution and then stored in phosphate buffer 10% formalin solution. The organs and tissues listed below (with the exception of the duodenum, jejunum, ileum, caecum, colon, rectum, ovaries, uterus, seminal vesicles, sternum, femur and individual identification parts) were embedded in paraffin and then sectioned. Hematoxylin-eosin (H.E.) staining was performed, and the samples from the 5 males and 5 females in the control- and high-dose groups which were used for the hematologic and blood chemistry studies were examined microscopically (in the cases of the bilateral organs, both organs were extracted and one was examined microscopically). A local effect of the administration of the test substance was observed in the stomachs; therefore, samples from 5 males and 5 females in the low- and medium-dose groups and the recovery groups were also examined microscopically, and typical examples of the normal and abnormal findings were photographed.

Organs examined: Cerebrum, cerebellum, pituitary gland, spinal cord (chest), sciatic nerve, thyroid, adrenals, thymus, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, lungs (including bronchi), stomach, duodenum, jejunum, ileum, caecum, colon, rectum, liver, kidneys, bladder, testes, epididymides, ovaries, uterus, seminal vesicles, sternum (including marrow), femur (including marrow), individual identification parts (earlobes).

Postmortem examinations (offspring):

SACRIFICE
On lactation day 4, all pups were sacrificed by exsanguination under ether anesthesia and autopsied. The presence or absence of abnormalities in the organs and tissues, including the heads, chests, and abdomens were investigated.

Statistics:

Chi-square tests (significance levels 0.05 and 0.01, two-tailed) with Yates continuous correction were performed on the mating rates, fertilization rates, conception rates, birth rates, sex ratios of surviving pups, auditory responses, approaching responses, touch responses, pain responses, pupil responses, and righting reflexes. In cases in which cells with expected frequencies of 5 or less were seen, tests were performed by Fisher’s direct probability calculation method (significance levels 0.05 and 0.01, two-tailed). For the implantation rates, stillbirth rates, live birth rates, external abnormality rates, and survival rates of live-born pups; after the rates were obtained for each animal, homoscedasticity was tested by Bartlett’s test. In the homoscedastic cases, Dunnett’s tests were performed, and in the nonhomoscedastic cases, Dunnett-type tests were performed (significance levels 0.05 and 0.01, two-tailed). For the other numerical results, the uniformities of the dispersions of the various groups were first tested by Bartlett’s method (significance level 1%, two-tailed). When the result was a uniform dispersion, the differences of the mean values of the control and the various administration groups were tested using Dunnett’s method; when the results were non-uniform, the differences in the mean ranks of the control and the various administration groups were tested, using a Dunnett-type method (mean rank test method) (significance levels for both cases: 5% and 1%, two-tailed). Mann-Whitney U tests (significance levels: 5% and 1%, one-tailed) were performed for the qualitative urinalysis items and the results of the histopathological examinations.

Reproductive indices:

After the end of the pre-mating administration period, the males and females of the same principal administration groups were allowed to cohabit overnight and the females which were found to form vaginal plugs or in which semen were observed in the vaginal smears the next morning were considered to have mated. The number of days required until mating were calculated, with the day the mating started as day 0. The mating rates (%) [(number of mated animals/number of cohabiting animals) x 100], the conception rates (%) [(number of fertilized females)/number of cohabiting females) x 100], and the fertilization rates (%) [(number of fertilized females)/(number of cohabiting males) x 100] were calculated from the mating results. The numbers of corpora lutea and the numbers of implantatios were counted and the implantation rates (%) [(number of implantations)/(number of corpora lutea) x 100] were calculated.

Offspring viability indices:

The rate of still births (%) [(number of stillborn/total number of pups delivered) x 100], the birth rates [(number of surviving pups/total number of pups delivered) x 100], the liveborn survival rates (%) [(number of pups surviving on day 4 of lactation)/(number of live pups on day 0 of lactation) x 100] were calculated.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality during the study period. No effects on the clinical signs were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No statistically significant differences in body weight were observed between the control groups and the treatment groups.

A significant decrease in food consumption was seen on day 42 in the males administered 250 mg/kg bw/d only. A significant increase was noted at a few time points in females administered 1000 mg/kg bw/d in the main group (gestation day 4) and the satellite group (administration day 25 and 29, recovery day 11). These variation are not considered to be treatment-related, as they were transient and no effect on body weight was observed.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test substance was administered by gavage daily, with doses based on the body weight, ensuring an accurate dosing of the animals.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There was no difference in the number and duration of estrous cycles between the control group and dose groups (see table 1).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- All 12 pairs of each dose group copulated successfully (copulation index 100%) (see table 3 and 4). One pair in the control group mated on day 7 after the mating period started and the other pairs mated within 4 days.
- All females became pregnant (fertility index: 100)
-The gestation index was 100% for all dose groups
- The delivery index was 100% for all dose groups
- All females delivered live pups

All the females delivered normally on days 21.1–22.5 of pregnancy, and no significant differences were observed between the control group and the treatment groups with respect to gestation periods, numbers of corpora lutea, implantation rates, stillbirth rates, number of live pups delivered, or live birth indexes (see table 2a and b).


ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant increases in the absolute kidney weight of the females in the 500 and 1000 mg/kg bw/d groups was observed. However, as the increase was not dose-related and only observed in one sex, this effect is not considered to be an adverse effect. An increase in absolute thyroid weight of females in the exposed satellite group was observed, but probably not treatment-related as the effect was not observed in the main group and no histopathological findings were reported. A significant increase in the absolute spleen weight in females administered 500 mg/kg bw/d was deemed to be incidental and thus not treatment-related. There were no effects on reproduction organs in either sex. The ovary weights were not recorded.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects were observed on reproductive organs and tissues, nor on other organs and tissues.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The results of the main and satellite groups have been assessed together, as there were no major differences in the type of observed effects.
Effects on the forestomach and corpus in males was observed as slight/mild cellular infiltration, erosion and increased numbers of globular leucocytes, and thickening of the limiting ridge. The incidences and severity of the effects increased in the males in the 1000 mg/kg bw group, compared to the other groups. In the females, only slight/mild erosion of the corpus was observed in 1-2 animals in each dose groups. At the end of the recovery period, increased numbers of globular leukocytes in the corpus and thickening of the limiting ridge were still observed, but their severity had decreased, suggesting these effects are reversible. Although the repeated use of a stomach tube to administer the test substance may have caused some of the damage, the main cause of effects were probably the local irritating effect of the test substance. As the limiting ridge and forestomach is specific to the rat physiology, these results are not considered to be relevant to humans.

No systemic effects were observed and there were no effects on reproductive organs and tissues.

OTHER EFFECTS (PARENTAL ANIMALS)

HAEMATOLOGY
A statistically significant decrease in the eosinophil level in the males administered 1000 mg/kg bw/d during week 6 was observed. As the overall white blood cell count and percentage was unchanged compared to the control group and the other white blood cell parameters were within historical control ranges (Petterino, 2006*), this is not considered to be an adverse effect of the treatment, but rather a reversible variation. In the 1000 mg/kg bw satellite groups small, but statistically significant, decreases were observed during the recovery period. The RBC-, hemoglobin- and hematocrit values of the males were significantly reduced. The levels of other blood cell types were not affected. The levels of RBC, hemoglobin and hematocrit measured in the males administered 1000 mg/kg bw were higher than the levels measured in the control males in the main group, indicating that the normal range is broad. These values also fell within the historical control range for male Sprague-Dawley rats. The reticulocyte level of the females in the satellite group administered 1000 mg/kg bw/d was slightly, but statistically significantly reduced. The levels of other blood cell types were not affected. Therefore, the effects on animals in the highest dose group are not considered to be treatment-related.
Changes observed in the female 500 mg/kg group (statistically significant increase in lymphocyte percentage and statistically significant reduction in segmented neutrophil percentage) are considered to be incidental, as they were not dose-related.

*Petterino, C. and Argentino-Storino, A. Clinical chemistry and haematology historical data in control
Sprague-Dawley rats from pre-clinical toxicity studies. Experimental and Toxicologic Pathology 2006; 57: 213–219

CLINICAL CHEMISTRY
In males in all dose groups a statistically significant increase in the A/G ratio was observed. However, as no effect was seen in the absolute protein and albumin levels, this not considered to be toxicologically relevant. In the males in the 1000 mg/kg bw/d group, significant increases in the triglycerides and urea nitrogen, as well as a significant reduction in the chlorine value were observed. The females of the same dose group showed a significant decrease in the gamma-GTP level, although the actual mean value was the same as for the control group. These effects may be treatment-related, but are not considered to be adverse effects, as only one sex was affected and no other effects on clinical chemistry parameters or histopathology were reported. No effects were observed in the satellite groups.

URINALYSIS (males)
There were no significant differences between the males in the control group and the treatment groups. However, a tendency towards increased pH in the urine with increasing doses was noted. The pH had normalised in the satellite group during the second week of recovery. This indicates that the test substance causes a higher urinary pH in exposed rats, but this effect is not considered to be adverse.

NEUROBEHAVIOUR
A statistically significant decrease in the number of rearings was observed in the males in the 1000 mg/kg bw/d main group in the open field tests during week 3, 4, 5 and 6 of the study. In contrast, a statistically significant increase in the number of rearings was observed in females in the satellite group administered 1000 mg/kg bw/d during week 5 of the study. The effect was just seen in either the main or satellite group and there was a divergence in effect between the sexes; furthermore, no effect was seen on other parameters. The overall result does therefore not indicate the test substance causes adverse neurological effects.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

VIABILITY (OFFSPRING)
No significant differences were observed in viability of the pups between the control groups and the treatment groups. The viability on day 4 was similar in all groups (see table 2b).

BODY WEIGHT (OFFSPRING)
The body weight of the males in the 250 mg/kg bw/d group was significantly increased, compared to that of the control group, at the time of birth. As it was not dose-related, only observed in one sex and at one time point, it is not considered to be an adverse effect. No significant differences in body weights were observed between the control groups and the treatment groups on lactation day 4 (see table 5).

GROSS PATHOLOGY (OFFSPRING)
One live-born pup in the 1000 mg/kg bw/d group was born with a vestigial tail, however, due to the single incidence, this was not considered to be related to the treatment with the test substance. Apart from this, no unusual findings were noted during the gross necropsy of the pups that survived until lactation day 4.

OTHER FINDINGS (OFFSPRING)
A statistically significant difference in the sex ratio (No. males/No. liveborn pups) was observed in the 500 mg/kg bw/d group (0.44 versus 0.58 in the control group). As this effect was not seen in any other dose groups, it is not considered to be treatment-related (see table 5).

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

Reproductive effects observed:

not specified

Table 1: Estrous cycle in female rats during pre-mating period (main group)

		Estrous count
Dose (mg/kg bw)	No. animals	0	1	2	3	4	Mean ± SD	Mean duration of cycles Mean ±SD
0	12	0	0	0	5	7	3.6 ± 0.5	4.1 ± 0.3
250	12	0	0	0	5	7	3.6 ± 0.5	4.3 ± 0.4
500	12	0	0	0	1	11	3.9 ± 0.3	4.1 ± 0.1
1000	12	0	0	0	4	8	3.7 ± 0.5	4.1 ± 0.3

 

Table 2a: Delivery data on females in all groups

Dose (mg/kg bw)		No. pregnant females	Gestation period	No. of corpora lutea	No. of implantations	Implantation index
0	Total Mean SD	12	22.0 0.4	207 17.3 1.5	192 16.0 1.5	92.9 5.4
250	Total Mean SD	12	22.3 0.3	200 16.7 2.1	188 15.7 2.2	94.0 5.1
500	Total Mean SD	12	22.1 0.3	209 17.4 1.5	199 16.6 1.9	95.1 5.1
1000	Total Mean SD	12	22.2 0.4	201 16.8 2.2	193 16.1 1.8	96.4 5.9

 

Table 2b: Delivery data on females in all groups

Dose (mg/kg bw)		No. pregnant females	No. of stillborn (%)	No. of live born	Live birth index %	Viability index day 4 after birth
0	Total Mean SD	12	3 (1.5) (3.8)	183 15.3 1.3	98.5 3.8	97.9 3.1
250	Total Mean SD	12	1 (0.6) (1.9)	171 14.3 3.0	99.4 1.9	97.0 4.8
500	Total Mean SD	12	2 (1.0) (2.4)	178 14.8 2.9	99.0 2.4	98.1 2.9
1000	Total Mean SD	12	2 (1.1) (2.7)	182 15.2 1.7	98.9 2.7	98.0 5.3

Table 3: Mating and fertility data, males

 

Dose (mg/kg)	N	Days until copulation Mean ± SD	Copulation index (%)*	Insemination index (%)**
0	12	2.7±1.7	12/12 (100)	12/12 (100)
250	12	2.6±1.2	12/12 (100)	12/12 (100)
500	12	2.4±0.9	12/12 (100)	12/12 (100)
1000	12	2.8±1.1	12/12 (100)	12/12 (100)

* (No. of copulated animals/No. of mated animals) x 100

** (No. of males that inseminated females/No. of copulated females) x 100

 

 

Table 4: Mating and fertility data, females

 

Dose (mg/kg)	N	Days until copulation Mean ± SD	Copulation index (%)*	Fertility index (%)**
0	12	2.7±1.7	12/12 (100)	12/12 (100)
250	12	2.6±1.2	12/12 (100)	12/12 (100)
500	12	2.4±0.9	12/12 (100)	12/12 (100)
1000	12	2.8±1.1	12/12 (100)	12/12 (100)

* (No. of copulated animals/No. of mated animals) x 100

** (No. of pregnant animals/No. of copulated females) x 100

Table 5: Pup data

 

Dose (mg/kg bw)		Sex ratio	Body weight males	Body weight females
0	Total Mean SD	0.58	6.4 0.5	6.2 0.4
250	Total Mean SD	0.54	6.9* 0.5	6.5 0.5
500	Total Mean SD	0.44*	6.7 0.5	6.3 0.4
1000	Total Mean SD	0.55	6.7 0.4	6.3 0.4

* p<0.05

Conclusions:

AEPD had no effect on the reproductive performance.

Executive summary:

A combined repeated dose and reproduction/developmental toxicity screening study performed according to OECD 422 to assess the potential reproductive and fertility effects of 2-amino-2-ethyl-1,3-propanediol (AEPD) is available (Ishida, 2004). In this study doses of 250, 500 and 1000 mg/kg bw/day of the test substance were administered by gavage to rats during pre-mating, mating, gestation and until lactation day 4. A satellite group with a recovery period of 14 days after dosing ended was included in the control and 1000 mg/kg bw/day groups. As no effects were observed up to and including the highest dose level, the NOAEL for systemic toxicity is considered to be>1000 mg/kg bw/day. In the P-generation, the parental reproduction parameters (including mating index, fertility index, gestation period, delivery index, and number of live pups) were not affected by treatment with the test substance up to and including the highest dose level of 1000 mg/kg bw/day. The NOAEL for reproduction is therefore considered to be >1000 mg/kg bw/day.

Reference 2

Endpoint:

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

There are no animal data available on the developmental toxicity of 2-amino-2-ethyl-1,3-propanediol (AEPD) according to OECD 414. In a combined repeated dose and reproduction/developmental toxicity screening study with AEPD according to OECD 422, no adverse effects on reproduction parameters (including mating index, fertility index, gestation period, delivery index, and number of live pups) and on viability in the F1 generation were observed by treatment with AEPD (Ishida, 2004). In this study doses of 250, 500 and 1000 mg/kg bw/day of the test substance were administered by gavage to rats during pre-mating, mating, gestation and until lactation day 4. A satellite group with a recovery period of 14 days after dosing ended was included in the control and 1000 mg/kg bw/day groups. As no effects were observed up to and including the highest dose level, the NOAEL for systemic toxicity is considered to be > 1000 mg/kg bw/day. In the pups (F1-generation), no effects on viability were observed. The body weight of male pups in the 250 mg/kg bw/day group was significantly increased, compared to that of the control group, at the time of birth. As it was not dose-related, only observed in one sex and at one time point, it is considered to have no biological significance. No treatment-related histopathological findings were noted at study termination. A statistically significant difference in the sex ratio (no. males/no. live-born pups) was observed in the 500 mg/kg bw/day group (0.44 versus 0.58 in the control group). As this effect was not seen in any other dose groups, it is not considered to be treatment-related. Therefore, the NOAEL for teratogenicity was set to be > 1000 mg/kg bw/day.

 

Other data are available for other members of the chemical category to which AEPD belongs. Therefore, read-across was performed based on a category approach.

The members of the category of 2-amino-1,3- propane-diols are substances that share a common propane backbone with an amine group at 2-carbon position and primary alcohols at 1 and 3 positions. The following substances are thus members of the aminopropanediol category: 2-amino-2-ethyl-1,3-propanediol (AEPD, CAS No. 115-70-8), 2-amino-2-methyl-1,3-propane-diol (AMPD, CAS No. 115-69-5), 2-amino-1,3-propanediol (APD, CAS No. 534-03-2) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (trometamol, CAS No. 77-86-1) The only structural difference between trometamol and AEPD is a replacement of a hydroxyl group with a methyl group. Further analogues differ in the length of the alkyl side-chain at position 2 so that the following sequence is obtained: from 0 carbon atoms (APD) through 1 (AMPD) to 2 (AEPD). There are no other functional groups present in these molecules.  

 

The modelling of potential metabolites via the OECD QSAR toolbox v.2.0 (2010) did not predict relevant metabolites of the category members. Based on the chemical structure of the parental compounds, no metabolism is expected. Therefore, it can be assumed that aminopropanediols will not show reactive properties under in vitro and in vivo test conditions. All the category members are of low concern with regard to systemic toxicity. Available studies via the oral, dermal and intraperitoneal route indicate low acute and repeated dose toxicity. Inhalation is of no concern, because the low vapour pressure means that exposure is unlikely to occur. The results of the acute studies, as well as the repeated dose studies, demonstrate that the main cause of toxicity was the intrinsic alkalinity of the category members at the site of contact. The Cramer classification (related mainly to the oral route) also indicates a low toxicological concern for all the category members. No metabolism by cytochrome P450 enzymes in vivo is expected; this is supported by predictions from QSAR modelling.

 

With respect to reproduction and developmental toxicity, there is an in vitro limb bud micromass assay and a developmental toxicity screening study in rats available with AMPD. Embryotoxicity was assessed in thein vitrolimb bud micromass assay by exposing undifferentiated rat embryo limb bud mesenchymal cells to AMPD concentrations of 0.01 to 1000 µM in the cell culture medium (Ellis-Hutchins and Marshall, 2011). Following cell differentiation into chondrocytes, specific parameters (cell differentiation, cell viability, cell number, neutral red uptake, cell growth) were determined. Under the scope of the assay, the results predict a lack of developmental toxicity for AMPD.

 

In a non-guideline developmental toxicity screening study, the potential of AMPD to cause developmental toxicity in rats was investigated (Rasoulpour and Andrus, 2011). Female rats were administered AMPD from before mating until gestation day 14. During the pre-mating period the groups were administered either increasing doses from 100 up to 1000 mg/kg bw/day (34 days) or 1000 mg/kg bw/day, the limit dose (6 days). The rats were sacrificed on gestation day 14 and number and position of implantations, viable embryos, and resorptions were recorded. In addition the number of corpora lutea were counted and uteri of females lacking visible implantations were examined for signs of pregnancy (early resorption). No differences were observed between the control group and the treatment groups regarding numbers of corpora lutea, implantation rates, resorption rates, pre-implantation loss, post-implantation loss and number of normal embryos per litter. The examination of the reproductive tract did not reveal treatment-related effects on the number and position of implantations, viable embryos, resorptions or ovarian corpora lutea.

 

In order to meet the standard information requirements according to Regulation (EC) 1907/2006 Annex IX, Column I, 8.7.2, a GLP-compliant prenatal development toxicity study according to OECD 414 is required. Due to the structural similarity between the members of the category and the similar toxicological properties, APD, AMPD and AEPD form a robust chemical category and read-across within the chemical category is justified. Based on the information that APD represents the basic molecular structure compared with AMPD and AEPD, which contain an additional methyl or ethyl group, APD has been selected as the most appropriate and representative category member. APD is characterised by, as all other members, the presence of a propane backbone with three functional groups substituted.

 

Therefore, a prenatal developmental toxicity study following OECD 414 in rats via the oral route has been conducted with the category member APD. The test item 2-aminopropane-1,3-diol (APD) was administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19). There was no test item effect on maternal body weight / body weight gain, corrected body weight /body weight gain and on food intake in any test item groups up to and including 1000 mg/kg bw/day. There were no toxicologically significant differences, or test item related-changes in the evaluated intrauterine parameters (including number of corpora lutea and implantation site, pre- and post-implantation loss, number of foetal death and total intrauterine mortality) up to and including 1000 mg/kg bw/day. No remarkable test item-related internal or external observations were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups. No toxicologically relevant adverse effect of the test item was observed on the foetal parameters (number of viable foetuses, sex distribution of foetuses, mean foetal weight per litter, number of foetuses with retarded body weight) up to and including 1000 mg/kg bw/day. There was no biologically relevant and/or statistically significant increase in external, visceral and/or skeletal malformations in any test item treated groups when compared to control. Some skeletal variations ascribed to slightly retarded ossification (unossified hyoid body on the skull, ossified sternebra (3 or less); unossified vertebra and carpal, ossified ≤ 2.5) showed higher incidence (mostly with statistical significance) in the high dose and mid group. These variations are all associated with delayed ossification at skeletal development, commonly related to retarded foetal development. No signs of maternal toxicity were detected during the study based on clinical signs and bodyweight; however, in the 90-day rat repeated dose toxicity study with this test item, systemic toxicity was observed, in the absence of body weight or clinical signs. The foetal skeletal findings were considered to reflect a non-adverse, slight retardation of ossification. In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses other than skeletal variations related to delayed ossification during skeletal development. The No-Observed-Adverse-Effect Levels (NOAEL) for maternal toxicity, embryotoxicity, foetotoxicity, foetal malformations and skeletal development were determined to be 1000 mg/kg bw/day. The No-Observed-Effect Level (NOEL for skeletal development was 100 mg/kg bw/day

 

Literature not cited in the IUCLID :

1. Ellis-Hutchins and Marshall, 2011, ANGUS MOLECULES (DMAMP, AMPD AND 3-AB): IN VITRO EMBRYOTOXICITY SCREENING STUDY IN THE RAT EMBRYO LIMB BUD MICROMASS ASSAY, Report no. 100118, Company study no. DR-0050-0118-010

2. Rasoulpour and Andrus, 2011, ANGUS MOLECULES (DMAMP, AMPD AND 3-AB): IN VIVO DEVELOPMENTAL TOXICITY SCREENING STUDY IN Crl:CD(SD) RATS, Report no. 100176, Company study no. DR-0050-0118-011

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

April-November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The justification for category approach/read-across has been attached in section 13 of this IUCLID. In the enclosed document arguments are given for a category approach for four 2-amino-1,3-propane-diols. These substances share a common propane backbone with an amine group at 2-carbon position and primary alcohols at 1 and 3 positions. The members of the aminopropanediol category are: 2-amino-1,3-propanediol (APD, CAS No. 534-03-2), 2-amino-2-methyl-1,3-propane-diol (AMPD, CAS No. 115-69-5), 2-amino-2-ethyl-1,3-propanediol (AEPD, CAS No. 115-70-8), and 2-amino-2-(hydroxymethyl)-1,3-propanediol (trometamol, CAS No. 77-86-1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Name: 2-Aminopropane-1,3-diol
Other name: Serinol, APD (Aminopropanediol)
Molecular formula: C3H9NO2
CAS number: 534-03-2
Batch number: F100G6RR10
Appearance: White solid
Purity: 100%
Manufacturer: ANGUS Chemical Company
Manufacture date: 14 January 2016
Expiry date: 27 June 2017
pH: 11

Storage conditions: Room temperature, under inert gas (15-25 Celsius, below 70 RH%)
Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials will be applied to assure personnel health and safety. Dangerous to the environment.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Species and strain: Hannover Wistar rats (CRLHan)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
Housing condition: Standard laboratory conditions; individual housing
Target age of animals: Young adult female rats, nulliparous and non-pregnant, at least approximately 11 weeks old
Target body weight: Will not exceed ± 20% of the mean weight at onset of treatment
Acclimatisation period: At least 5 days

Animal health: Only healthy animals will be used for the test, as certified by the staff veterinarian.
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany), or similar laboratory bedding suitable for the purposes of the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents will be housed individually. Nest building material (ARBOCEL natural crinklets produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, Rosenberg, D-73494 Germany), or similar will be also added to the cages – the certificate of analysis will be included in the report) will be added to the cages.

The temperature and relative humidity values were measured twice daily during the acclimatisation period and during the experiment.

The animals will be provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water as for human consumption, ad libitum. The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier will provide an analytical certificate for the batch used.

The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis will be included in the raw data, and will be archived at CiToxLAB Hungary Ltd.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd. During the formulation process, the pH of each formulation will be adjusted using 10% HCl solution to achieve a pH<10, this fact will be documented in the raw data and reported.ormulations were prepared fresh prior to administration to animals or at appropriate frequency to allow their use according to stability assessments. Stability of the 2-aminopropane-1,3-diol in the vehicle (distilled water) was assessed in the
conditions employed on the study during the validation study.

The analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples will be taken from the test item formulations, each analysed at least two times during the study (approximately during the first and last week of treatment). Similarly, one sample will be taken in duplicate on each occasion from the middle of the control (vehicle) formulation for concentration measurement.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. The contentd of 2-aminopropane-1,3-diol and homogeneity of the dosing formulations was determined on two analytical occasions during the test. Analytical samples were taken from each test concentration to determine concentration and homogeneity and from the vehicle control. The samples were derivatized with trifluoroacetic acid diluted into the calibrated range with water then analysed by GC method.
.

Details on mating procedure:

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimatisation the females, according to their oestrus cycle, were paired with males for approximately 2 hours (1 male: 1-3 females) in the morning, until at least 24 sperm-positive females/group are obtained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm-positive females were separated and caged individually.

The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day.

Duration of treatment / exposure:

The control or test item dose formulations was be administered to mated, spermpositive assumed pregnant female rats daily by oral gavage on a 7 days/week basis from Gestation Day (GD) 6 to GD19.

Frequency of treatment:

Daily from GD 4 till GD 19.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Actual dose: 92 mg/kg bw

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Acutal dose: 294 mg/kg bw

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Actual dose: 940 mg/kg

No. of animals per sex per dose:

24 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels were set based on available data, including the results of a Dose Range Finding (DRF) study by oral gavage in rats and another DRF study in pregnant rats, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg/kg bw/day) and a NOAEL at the mid dose (300 mg/kg bw/day) or at the lowest dose (100 mg/kg/bw/day). In the pregnant DRF study, no signs of maternal toxicity, embryotoxicity or foetotoxicity were observed in any test item treated dose groups (up to and including 1000 mg/kg bw/day). Based on these results, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study. The oral route is a potential route of human exposure to the test item and is considered suitable to provide the exposure required for this developmental toxicology study. A constant volume of 10 mL/kg bw was administered to all dose groups, including the controls. The individual volume of the treatment wasl be based on the most recent individual body weight of the animals.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

CLINICAL OBSERVATIONS: Yes
Clinical observations were made twice daily (at the beginning and end of each working day, or before treatment and when the peak of the clinical observations, if any, is observed after treatment). Only one clinical observation was made in the afternoon on those days when detailed clinical observation was made in the morning. Furthermore, clinical observations were made only once on necropsy days. Detailed clinical observations were made on all animals at the onset of treatment (GD6) then at least weekly and on the necropsy days. Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included, but were not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also be recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. On GD13 and/or 14 the sperm-positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

BODY WEIGHT: Yes
Body weight of each animal was recorded on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION: Yes
Food was measured on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD 6-20 and GD 0-20.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Before expected delivery, on GD 20, Caesarean section was performed on each treated dam. Sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia (pentobarbital sodium for injection.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; stomach of all animals (which will be examined thoroughly) and all the gross findings will be retained in 10% buffered formalin solution (or modified Davidson fixative, in case of the eyes, if any abnormalities noted) for possible future evaluation. The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and held in front of a light source to clearly identify the implantation sites. Uteri that appear non-gravid were further examined to confirm the non-pregnant status (i.e. by ammonium sulphide staining or a suitable alternative method). The corrected body weight was calculated (body weight on GD20 minus weight of the gravid uterus). The number of corpora lutea in each ovary and of implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted; the number and percent of pre- and postimplantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each live foetus were weighed individually and subjected to external examination, plus an additional examination of the great arteries. The gender of foetuses was determined according to the anogenital distance. Thereafter, the foetuses were individually identified; approximately half of each litter were subjected to visceral examination, and the other half will be processed for skeletal examination. For the foetuses subjected to visceral examination, the abdominal and thoracic region was opened, and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomya mixture: then after fixation, the body will be micro-dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. For the foetuses subjected to skeletal examination, the abdominal region was opened, and the viscera and skin of foetuses were removed; the cadavers were fixed in Alcian-blue -acetic acid-ethanol mixture. After fixation in isopropanol, the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope. All abnormalities (external, soft tissue and skeletal malformations, and variations) found during the foetal examinations were recorded.

Statistics:

Data were collected using the software PROVANTIS v.9, or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the software Microsoft Office Word and/or Excel, as appropriate. Data were collected to provide information on parameters including:

MATERNAL DATA:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals, no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day), if any
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD20 (body weight on GD20 minus gravid uterine weight) and corrected body weight gain (body weight gain of GD0-20 minus
gravid uterine weight): mean ± S.D.
- Net body weight change (body weight gain of GD6-20 minus gravid uterine weight): mean ± S.D.
- Gravid uterine weight: mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings,

CAESAREAN SECTION AND NECROPSY DATA:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live foetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D. Classified according to time of death: preimplantation loss, postimplantation mortality, early and late embryonic as well as foetal death.

Indices:

- Preimplantation loss: %, group mean: Number of corpora lutea-Number of implantations x 100 / Number of corpora lutea
- Postimplantation loss: %, group mean: Number of implantations-Number of live foetuses x 100 / Number of implantations

Foetal Data:
- Sex distribution: %, group mean: Number of male (female) foetuses x 100 / Number of foetuses
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses
- Visceral abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses
- Skeletal abnormalities/litter: %, group mean: Number of foetuses with abnormality x 100 / Number of foetuses

The statistical evaluation of data was performed with the program package SPSS PC+4.0 (SPSS Hungary Ltd., Budapest, Hungary) or SAS v9.2 (when using
Provantis).

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related adverse effects or systemic clinical signs were noted in the treated animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related effect on body weight was observed in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day) when compared to control. No toxicologically significant changes were observed in the mean body weights of the Low, Mid or High dose groups when compared to control. Similarly, no statistically significant or biologically relevant differences were seen in the body weight gain, corrected body gain or net body weight gain values during the treatment period (GD 6-20) or entire study (GD 0-20) compared to the control value

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No biologically relevant changes were observed in the mean daily food consumption of dams in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) compared to the control value. The food consumption of the Low, Mid and High dose groups was comparable with the Control group, the sight differences in the daily mean food consumption of the High dose group during the different periods were not larger than 8.0% when compared to the control. There was no statistical significance and clear dose response, furthermore based on the absolute values those differences were considered as being non-test item related background variations.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. There was no statistically significant difference in the preimplantation loss of the test item treated groups when compared to the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in the post-implantation loss between the test item treated and control groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no statistically significant difference in foetal death in any test item treated groups compared to the control

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

No test item related observations were recorded for any evaluated animals in the study.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

other: no effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight of foetuses per litter in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the High dose group (1000 mg/kg bw/day), mean foetal weight per litter was significantly (p<0.05) higher than control value, but based on the biological relevance this fact was considered as a non-adverse test item related effect.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was comparable with the control mean in all test item treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The weight of foetuses per litter in the Low and Mid dose groups (100 and 300 mg/kg bw/day, respectively) did not differ significantly from the control mean value. In the High dose group (1000 mg/kg bw/day), mean foetal weight per litter was significantly (p<0.05) higher than control value, but based on the biological relevance this fact was considered as a non-adverse test item related effect. The total number of retarded foetuses (runts) as well as the number of affected litters was comparable to control in all test item treated groups indicating no test item effect on this parameter.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no recorded external variations in this study. One malformation was recorded in 1 out 228 foetuses in the high dose group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Most of the skeletal findings corresponded with the current study control / HC data, or were incidental findings without dose response. However, in case of some skeletal variations (see further in this section), test item related increased incidence was observed in the High and Mid dose group, but no effect was noted in the Low dose group. The total number of foetuses with skeletal malformations did not differ from the control in a biologically relevant way (there was no clear dose response and the litter based incidence can not be clearly differentiated from an incidental frequency). The total number of foetuses with skeletal variations was significantly (p<0.01) higher in the High (1000 mg/kg bw/day) dose group than in the Control group, slightly increased number without statistical significance was detected in the Mid dose group (300 mg/kb bw/day), while no similar difference was noted in the Low dose group (100 mg/kg bw/day). Consequently, the total number of intact foetuses was lower than control in the High dose group. These findings indicated a slight test item effect in the High dose group.

Visceral malformations:

no effects observed

Description (incidence and severity):

All of the visceral findings are consistent in general nature and incidence with the study concurrent control data or the existing historical control data or were incidental findings without dose response, therefore considered as not related to the test item treatment. Based on the visceral findings, the number of malformed / variant / intact foetuses were comparable with the control in the Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively).

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Most of the findings correspond to the study control data or have isolated occurrence that were considered incidental ascribed to individual variability. In the case of a few skeletal variations, an apparent increased incidence of changes in absolute numbers and percentile litter mean were observed with statistical significance in the high dose when compared to control (skull, hyoid body, unossified; ossified sternebra (3 or less); vertebra, unossified; carpal, ossified ≤ 2.5). These variations are all associated with delayed ossification at skeletal development. Based on these results the test item did not cause a higher incidence of malformations, but some skeletal variations in the high dose group, related to delayed ossification, appeared to be related to treatment.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed

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:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: sternum

skeletal: vertebra

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Conclusions:

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses other than skeletal variations related to delayed ossification during skeletal development. The No-Observed-Adverse-Effect Levels (NOAEL) for maternal toxoicity, embryotoxicity, foetotoxicity, foetal malformations and skeletal development were determined to be 1000 mg/kg bw/day. The No-Observed-Effect Level (NOEL) for skeletal development was 100 mg/kg bw/day.

Executive summary:

The test item 2-aminopropane-1,3-diol (APD) was administered daily by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19). There was no test item effect on maternal body weight / body weight gain, corrected body weight /body weight gain and on food intake in any test item groups up to and including 1000 mg/kg bw/day. There were no toxicologically significant differences, or test item related-changes in the evaluated intrauterine parameters (including number of corpora lutea and implantation site, pre- and post-implantation loss, number of foetal death and total intrauterine mortality) up to and including 1000 mg/kg bw/day. No remarkable test item-related internal or external observations were recorded for any pregnant animals during necropsy. No remarkable abnormalities were observed on the placentas in any examined groups. No toxicologically relevant adverse effect of the test item was observed on the foetal parameters (number of viable foetuses, sex distribution of foetuses, mean foetal weight per litter, number of foetuses with retarded body weight) up to and including 1000 mg/kg bw/day. There was no biologically relevant and/or statistically significant increase in external, visceral and/or skeletal malformations in any test item treated groups when compared to control. Some skeletal variations ascribed to slightly retarded ossification (unossified hyoid body on the skull, ossified sternebra (3 or less); unossified vertebra and carpal, ossified ≤ 2.5) showed higher incidence (mostly with statistical significance) in the high dose and mid group. These variations are all associated with delayed ossification at skeletal development, commonly related to retarded foetal development. No signs of maternal toxicity were detected during the study based on clinical signs and bodyweight; however, in the 90-day rat repeated dose toxicity study with this test item, systemic toxicity was observed, in the absence of body weight or clinical signs. The foetal skeletal findings were considered to reflect a non-adverse, slight retardation of ossification.

 

In this study, from the observations made in the dams and their foetuses, there were no changes on embryos or foetuses other than skeletal variations related to delayed ossification during skeletal development. The No-Observed-Adverse-Effect Levels (NOAEL) for maternal toxoicity, embryotoxicity, foetotoxicity, foetal malformations and skeletal development were determined to be 1000 mg/kg bw/day. The No-Observed-Effect Level (NOEL for skeletal development was 100 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Justification for classification or non-classification

The available data on the reproduction, fertility and teratogenic toxicity, and on effects on or via lactation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.