2-amino-2-ethylpropanediol

Endpoint summary

Description of key information

Oral (OECD 422 with AEPD), 42 days, rat: NOAEL (systemic) ≥ 1000 mg/kg bw/day

Oral (OECD 408 with APD), 90 days, rats: NOAEL (systemic) 250 mh/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

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 reported

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: 10 weeks
- Weight at study initiation: 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 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on oral 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

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 using 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

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
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/day groups. In addition, a satellite group of 5 animals per sex was included in the control and 1000 mg/kg bw/day 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/day for 2 weeks, no effects of the test substance were observed. 1000 mg/kg bw/day 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/day as the highest dose and 500 and 250 mg/kg bw/day 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/day groups.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

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:
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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

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 apolipoprotein (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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals in the main groups were sacrificed on the day after the final administration (day 42 for the males and day 4 of lactation for the females), while the animals in the satellite groups were sacrificed on the last day of the recovery period. 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 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: Yes
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).

Other examinations:

A number of reproduction parameters were measured, which have been summarised in 7.8.1. key, Nishimura, 2004, screening, 42d, rat, RL1.

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.

Details on results:

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

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences in body weight were observed between the control groups and the treatment groups (see table 1 and 2).

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

The test substance was administered by gavage daily, with doses based on the body weight, ensuring an accurate dosing of the animals.

HAEMATOLOGY
A statistically significant decrease in the eosinophil level in the males administered 1000 mg/kg bw/day 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/d 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/day 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/day 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 bw/day 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/day 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 serious effects on clinical chemistry parameters or histopathology were reported. No effects were observed in the satellite groups, also suggesting that any effects noted during the administration period were reversible.

URINALYSIS (males)
There were no significant differences between 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 highly alkaline 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/day 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/day 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.

ORGAN WEIGHTS
Statistically significant increases in the absolute kidney weight of the females in the 500 and 1000 mg/kg bw/day groups was observed. However, as the increase was not dose-related and the relative kidney weights were not significantly increased, 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/day was deemed to be incidental and thus not treatment-related. The ovary weights were not recorded. See table 1 and 2.

GROSS PATHOLOGY
No effects were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
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/day 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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

Critical effects observed:

not specified

Table 1, Mean body weight and selected mean organ weights, female main group

	Dose mg/kg bw/day	Body weight, g (g/100 g BW)	Thyroid (R+L), mg (mg/100 g BW)	Kidney, g (g/100 g BW)	Spleen, g (g/100 g BW)
Absolute	0	299	20.9	2.00	0.60
	250	305	19.6	2.18	0.62
	500	306	22.4	2.33*	0.73
	1000	317	20.0	2.36*	0.65
Relative	0	-	7.0	0.67	0.20
	250	-	6.4	0.72	0.20
	500	-	7.3	0.76	0.23*
	1000	-	6.3	0.74	0.21

*statistically significant, p<0.05

 

Table 2, Mean body weight and selected mean organ weights, female satellite group

	Dose mg/kg bw/day	Body weight, g (g/100 g BW)	Thyroid (R+L), mg (mg/100 g BW)	Kidney, g (g/100 g BW)	Spleen, g (g/100 g BW)
Absolute	0	281	15.9	2.09	0.53
	1000	294	20.4*	2.09	0.48
Relative	0	-	5.7	0.74	0.19
	1000	-	6.9	0.71	0.16

*statistically significant, p<0.05

 

Conclusions:

AEPD did not cause systemic toxicity following repeated oral exposure

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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: 27 June 2016
Expiry date: 27 June 2017
pH: 11

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:WI Wistar rats
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld
- Hygienic level: SPF at the supplier, standard laboratory conditions during the study
- Number of animals: 40 males + 40 females, 8 males + 8 females for replacement purpose (no replacement was performed)
- Age of animals: young adult rats, 7 weeks old at start of treatment
- Target body weight: Not exceeded ± 20% of the mean weight for each sex at onset of treatment. Males: 231–261g. Females: 183–214g.
- Acclimation period: 7-8 days

DETAILS OF FOOD AND WATER QUALITY
- Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice–breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Tap water from municipal supply, as for human consumption from a 500 ml bottles, ad libitum. The supplier provided analytical certificate for the batch used, which are archived with the study raw data.
- Water quality control analysis was 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 A.u.36., Hungary).
- The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Cage type: Type II polypropylene
- Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany suitable for the purposes of the study. Nest building material was also provided for animals (Arbocel crinkles natural). D
- Light: Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 19.8–28.5°C
- Relative humidity: 25–50%
- Ventilation: 15-20 air exchanges/hour
- Housing/Enrichment: Rodents were housed up to 2 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.
- The temperature and relative humidity were measured continuously and recorded twice daily during the study and acclimation

Route of administration:

oral: gavage

Details on route of administration:

The doses were selected by the Sponsor based on available information and results of 14-day study by oral gavage in rats performed at CiToxLAB – Hungary Ltd. Based on these results, the 1000 mg/kg bw/day dose was considered to be acceptable as top dose in the 90-day repeated dose study. The oral
route was selected as it is a possible route of exposure to the test item in humans.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The Test Item was formulated in distilled water at the appropriate concentrations according to the dose levels selected, in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared prior to administration to the animals according to stability assessment results. During the dose formulation the pH of each concentration was adjusted to below pH 10, the added volume of 10% HCl solution used for adjustment and the final pH value of the formulations were documented in the raw data. The pH range of formulations applied in the study was: pH 9.44–9.99.

VEHICLE
- Distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY OF DOSING FORMULATIONS
Stability of the dosing formulations under the conditions of use during and covering the range of concentrations and storage used has been assessed as part of the validation study 16/243-316AN. Results confirmed that the test item formulations in distilled water at concentrations 6 and 100 mg/mL were stable at room temperature for 7 days.

ANALYSIS OF DOSING FORMULATIONS
To verify the concentration of the test item in formulations, representative samples were taken and analysed from each concentration 4 times during the study (week 1, 5, 8 and week 13), one set to analyse (which could be collected in replicates as practical) and one set as a back-up. No confirmatory analysis was required. Similarly, one sample was taken in duplicate from the Group 1 (control).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Ten males and 10 females/group were treated daily for 90 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume (10 mL/kg bw) was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Ten males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were made on all animals outside the home cage in a standard arena at randomization, prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

BODY WEIGHT: Yes
Body weight was recorded with a precision of 1g at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then weekly, including on Day 90 (last treatment day) and prior to necropsy. Weekly body weight and body weight gain were reported.

FOOD CONSUMPTION :
The determination of food consumption was performed for all groups once a week. The remaining, non-consumed food was weighed weekly from Day 8 with a precision of 1 g. Daily food consumption were calculated for reporting purposes.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was conducted in all animals before treatment and in the control (Group 1) and high dose (Group 4) animals on Week 13. Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL ciclopentolato hydrochloride; Batch No.: 160515, exp.: May 2021) and “Humapent” (5 mg/mL ciclopentolate hydrochloride; Batch No.: 8680415, exp.: April 2017) into the conjunctival sac. The evaluation was performed by external examination and using a Gowlland ophthalmoscope.

HAEMATOLOGY: Yes
Haematology parameters and blood clotting times were evaluated in all animals euthanized at termination, except one animal from which no sample could be taken (including coagulation) due to a technical error:

CLINICAL CHEMISTRY: Yes
Clinical chemistry parameters were evaluated in all animals euthanized at termination, except 2 animals form which no sample could be taken due to technical error:

URINALYSIS: Yes
Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which were placed in metabolic cages. The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable.

NEUROBEHAVIOURAL EXAMINATION: Yes
Towards the end of the treatment period on Day 82-84, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive),
assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968). Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated. To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data. Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from the high dose and control group were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

IMMUNOLOGY: No

Sacrifice and pathology:

Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia. After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. In addition, bone marrow smears from the femur of each animal were prepared at necropsy (but not examined).

Statistics:

Data were collected using the software PROVANTIS v.9 or were recorded on the appropriate forms as per relevant SOPs, then tabulated using PROVANTIS v.9,
Microsoft Office Word and/or Excel, as appropriate. Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System). The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data: The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (oneway analysis of variance) test was carried out.
If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item related clinical signs recorded during the study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in the study. All animals survived until the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects observed on the animal body weights or body weight gain values during the study. All treated groups were comparable with the control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related adverse effects noted on animal food consumption under the conditions of this study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes compared to pre-treatment or/and the control were noted at ophthalmoscopy examination. All examined animals were found to be normal.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the high dose, there were test item related effects observed on the haematology parameters evaluated at the completion of the 90-day treatment which considered to be relatively small (<10% difference) but adverse. In high dose animals in both sexes the results indicated Test Item related slight anaemia, with statistically significantly lower RBC, HGB, Haematocrit. Also the Reticulocytes percentage was increased statistically significantly High dose (in high dose females without statistical significance) suggesting a compensated anaemia in the high dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item related effects on the clinical chemistry parameters evaluated at the completion of the 90-day treatment period in high dose males (calcium).In males and females, although calcium was statistically significantly higher in Mid dose groups of both sexes, the values were considered to be in the historical range so this was not considered to be an adverse effect of the test item. In the high dose for both sexes all animals were above the concurrent control range and the mean was slightly out of the historical control range, therefore this finding in the high dose was considered to be test Item related. In the absence of any associated changes in clinical pathology or tissues at histology, it is not clear if the change is adverse or adaptive. Other statistical differences tended to be within the historical control range and/or without a dose response, therefore the changes were not considered to be related to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were noted on the urinalysis parameters evaluated, which were considered to be related to the test item. The urinalysis parameters were comparable to the controls in all dose groups. Variations occurred and consisted of minor differences to controls, these were unrelated to treatment and within the normal range.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test Item related findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli. There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity. Statistically significant difference was recorded for Mid and High dose females for the total travelled distance, during two five-minute intervals (at 30-35 min. and at 55-60 min).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Terminal body weights of animals were not statistically significantly different between the groups. When compared to the controls, the following changes in the liver,epididymis and kidney were observed in the mean organ weights and considered to be test item-related. The absolute and relative (to body and brain) liver weights were statistically significantly higher in high dose male and female groups. These differences were supported by test item-related histopathological findings noted in high dose rats discussed below; the weight difference at the High dose was attributed to treatment. Mid fose females had also statistically larger livers, but only when related to the bodyweight, however the values were all well within the historic control range and there were no histological changes to support this as being related to treatment. The relationship of the small difference in liver weight at the mid dose level is not considered to represent an adverse effect of treatment.

The absolute and relative (to body and brain) kidneys weights were statistically significantly higher in high dose males, and in mid and high dose females. These differences were supported by test item-related histopathological findings observed in high dose rats in both sexes, discussed below. In high dose males, the absolute kidney weights were larger by 14%, by 10% related to body and by 11% brain related. In the high dose females, absolute kidney weights were higher up by 20%, by 14% relative to body and by 23% brain related. At the female mid dose, although the kidney weights were statistically higher than concurrent controls, when compared with historic control data the weights were within the normal range. Furthermore, histologically the mid dose kidneys were normal hence the statistical weight difference is not considered to represent an adverse effect of the test item.

The absolute and relative (to body and brain) epididymis weights were statistically significantly lower at high dose. The absolute weights were lower by -12% and up to -15% relative to body and brain. However, there were no microscopic changes corresponded with these differences. The relationship between these statistical differences and high dose level of treatment is equivocal, and the values were all well within the historical range. Although, there was evidence of test item-related minimal to slight vacuolation seen by light microscopy in epididymis examined from high dose males, this change was unlikely to have contributed to the relatively small decrease of epididymis weights. Taking account of the historic data for epididymis weights and the lack of any histological evidence of a test item effect, it is considered that the statistical difference is not treatment related, and that all treated groups in this study had normal epididymis.

There were no other statistically significant differences considered as test item-related among groups in the weights of other organs measured when compared to controls. Statistical differences in adrenal weights were not considered to be treatment related; high dose males at the high dose had a mean very close to the overall historic mean value for adrenals, and the female miid dose mean was higher but without a dose response and was within the historic range. Since all weight values were considered normal and no test item-related microscopic findings were seen, these statistical differences were regarded as non-treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related dark/red multifocal discolorations of the glandular stomach mucosa were visualized at necropsy in 7/10 males dosed at 1000 mg/kg bw/day. These changes correlated with erosions/ulcers microscopically observed. Other changes were incidental or background.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Adverse test item-related findings were observed only in the stomach in animals at 1000 mg/kg bw/day, ascribed to a local irritation effect. In the liver, kidney and epididymis of animals at 1000 mg/kg bw/day minimal to slight changes were noted but were not considered to be adverse. No histological test item-related changes were noted in organs examined from the animals dosed at 250 mg/kg bw/day.

LIVER
Minimal to slight hepatocellular hypertrophy (centrilobular) altered liver in 4/10 high dose males. Minimal severity was observed in 6/10 high dose females. Enlarged cytoplasm of hepatocytes, with granular appearance of hepatocyte cytoplasm (without degenerative features) were characteristic microscopic features. This change was considered to be an adaptive non-adverse effect.

KIDNEY
Minimal to slight bilateral/unilateral focal/multifocal tubular vacuolation in the cortex/outer strip (medulla) was seen in 4/10 males and 2/10 females from the high dose groups. This change was considered to be non-adverse, since was not associated with any degenerative/necrotic or inflammatory findings.

EPIIDIDYMIS
Minimal to slight bilateral vacuolation of the epithelial cells in the caput (proximal) was recorded in 9/10 High Dose males. These minimal or slight changes appeared without any accompanied degeneration/necrosis or inflammation, as well as without evidence of abnormal sperms seen by light microscopy. Therefore, the vacuolation was considered to be non-adverse.

STOMACH (glandular)
Minimal to slight focal/multifocal erosion/ulcer (with congestion/haemorrhage) of the fundus/pylorus was present in 5/10 males and 1/10 female at the high dose level. The loss of mucosa with partial mucosal penetration without associated inflammatory response was microscopically seen. These lesions were considered as local irritant adverse effects. Other changes were incidental or background.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no adverse or yest item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

cauda epididymis

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

Under the conditions of this study, there were no effects on clinical signs or body weight. Test item related adverse effects were observed after 90 days of treatment in the high dose group; effects included signs of erosion/ulcer in the stomach. There were test item related higher liver, epididymis and kidney organ weights at the high dose in both sexes. At the high dose level only, minor histological changes in the liver, kidney and epididymis were not considered to be adverse, and slightly high serum calcium was observed. There were no adverse effects of test item in the mid or low dose groups of either sex. The NOAEL (no observed adverse effect level) was considered to be 250 mg/kg bw/day.

Executive summary:

The purpose of this study was to obtain information on the toxicity of 2-aminopropane-1,3-diol when administered daily for 90 days by oral gavage to the rat at 3 dose levels, based on previous data available, including the results of a 14 -day DRF study by oral gavage in rats. Male and female Wistar rats were treated once daily for 90 days by oral gavage administration. The dose levels used were 0, 62.5, 250 and 1000 mg/kg bw. Control animals were treated with the vehicle (distilled water).

There was no death and no test item related clinical signs during the study. There were no findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. There were no test item related effects observed on the animal body weights or body weight gain and food consumption values during the study. All examined animals were found to be normal during the ophthalmoscopy examinations. Haematology parameters evaluated at the completion of the 90-day treatment period showed adverse effects or findings that were test item related. In high dose animals in both sexes the results indicated test item related slight anaemia, with statistically significantly lower RBC, HGB, Haematocrit. Also the reticulocytes percentage was increased statistically significantly in the high dose group (in high dose females without statistical significance), suggesting a compensated anaemia in the high dose group. There was a slight test item related effect with apparently increased serum calcium at the high dose in both sexes. There were no adverse test item related effects on the urinary parameters evaluated at the completion of the treatment period. There were no adverse or test item related observations in the animal oestrus cycle

evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases. Test item-related dark/red multifocal discolorations of the glandular stomach mucosa were visualized at necropsy in 7/10 males dosed at 1000 mg/kg bw/day. These changes correlated with erosions/ulcers microscopically observed. These observations were compatible with the slight, compensated anaemia which is common in the event of stomach ulcers. There were test item related higher liver, epididymis and kidney organ weights at the high dose in both sexes. Microscopic evaluation identified minor test item-related findings in the stomach, liver, kidney and epididymis in animals at 1000 mg/kg bw/day. No test item-related changes were noted in these organs examined from the animals dosed at 250 mg/kg bw/day. Minimal to slight erosion/ulcer of the stomach was present at the high dose level, these lesions were considered as local irritant adverse effects. Minimal to slight hepatocellular hypertrophy in the high dose was considered to be an adaptive non-adverse effect. Minimal to slight tubular vacuolation was seen in less than half the animals at the high dose groups; this change was considered to be non-adverse, since it was not associated with any degenerative/necrotic or inflammatory findings. Minimal to slight vacuolation of the epithelial cells in the caput (proximal) were observed without any accompanied degeneration/necrosis or inflammation, there was no evidence of abnormal sperms by light microscopy, therefore, the vacuolation was considered to be non-adverse.

In conclusion, under the conditions of this study, test item related adverse effects were observed after 90 days of treatment in the high dose group; effects included signs of anaemia and erosion/ulcer in the stomach. There were test item related higher liver,

epididymis and kidney organ weights at the high dose in both sexes. There were no adverse effects of test item in the mid and low dose groups of either sex. The NOAEL (no observed adverse effect level) was considered to be 250 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD 422 study with AEPD

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A combined oral repeated dose toxicity study and reproduction screening test was performed with 2-amino-2-ethyl-1,3-propanediol (AEPD), according to OECD 422 (Ishida, 2004). Rats (12/sex/dose) were administered 250, 500 and 1000 mg/kg bw/day of the test substance by gavage, with a satellite group of 5 animals per sex included in the control and 1000 mg/kg bw/day groups that was kept until 14 days after exposure ended to observe any late effects. Males were exposed for 42 days and females for 42-48 days (from 14 days before mating until day 4 of lactation). There was no mortality during the study period and no effects on the clinical signs or body weight were observed. In male animals of the high dose group, isolated decreases in haematological parameters were observed. As the effects occurred in males only, they are not considered to be treatment related. Moreover, the effects were not observed in the main and satellite group. Some clinical chemistry parameters were also changed compared to the concurrent controls in high dose animals. These values fell within the historical control values and are therefore considered not to have a biological significance. Except for incidental changes in kidney weights in females in the 500 and 1000 mg/kg bw/day groups, no effects were observed. No effects were observed on the gross pathology. The histopathological examinations showed reversible effects in the forestomach and corpus in treated animals. The main cause of these effects was most probably local irritation due to the highly alkaline test substance (pH 11.18 in 1% solution) administered by gavage. Therefore, the NOAEL is > 1000 mg/kg bw/day for males and females in this study.

In order to meet the standard information requirements according to Regulation (EC) 1907/2006 Annex IX, Column I, 8.6.2, a GLP-compliant sub-chronic toxicity study according to OECD 408 study is required. As no sub-chronic data are available with AEPD, a 90-day repeated dose toxicity study following OECD 408 in rats via the oral route has been performed with the category member 2-amino-1,3-propanediol (APD), and read-across within the chemical category is considered appropriate. 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.

A 90-day repeated dose toxicity, oral gavage study is rats was conducted with the category member 2-aminopropane-1,3-diol (APD). The dose levels were based on previous data available, including the results of a 14-day DRF study by oral gavage in rats. Male and female Wistar rats were treated once daily for 90 days by oral gavage administration. The dose levels used were 0, 62.5, 250 and 1000 mg/kg bw. Control animals were treated with the vehicle (distilled water). There was no death and no test item related clinical signs during the study. There were no findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. There were no test item related effects observed on the animal body weights or body weight gain and food consumption values during the study. All examined animals were found to be normal during the ophthalmoscopy examinations. Haematology parameters evaluated at the completion of the 90-day treatment period showed adverse effects or findings that were test item related. In high dose animals in both sexes the results indicated test item related slight anaemia, with statistically significantly lower RBC, HGB, Haematocrit. Also the reticulocytes percentage was increased statistically significantly in the high dose group (in high dose females without statistical significance), suggesting a compensated anaemia in the high dose group. There was a slight test item related effect with apparently increased serum calcium at the high dose in both sexes. There were no adverse test item related effects on the urinary parameters evaluated at the completion of the treatment period. There were no adverse or test item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases. Test item-related dark/red multifocal discolorations of the glandular stomach mucosa were visualized at necropsy in 7/10 males dosed at 1000 mg/kg bw/day. These changes correlated with erosions/ulcers microscopically observed. These observations were compatible with the slight, compensated anaemia which is common in the event of stomach ulcers. There were test item related higher liver, epididymis and kidney organ weights at the high dose in both sexes. Microscopic evaluation identified minor test item-related findings in the stomach, liver, kidney and epididymis in animals at 1000 mg/kg bw/day. No test item-related changes were noted in these organs examined from the animals dosed at 250 mg/kg bw/day. Minimal to slight erosion/ulcer of the stomach was present at the high dose level, these lesions were considered as local irritant adverse effects. Minimal to slight hepatocellular hypertrophy in the high dose was considered to be an adaptive non-adverse effect. Minimal to slight tubular vacuolation was seen in less than half the animals at the high dose groups; this change was considered to be non-adverse, since it was not associated with any degenerative/necrotic or inflammatory findings. Minimal to slight vacuolation of the epithelial cells in the caput (proximal) were observed without any accompanied degeneration/necrosis or inflammation, there was no evidence of abnormal sperms by light microscopy, and he vacuolation was therefore considered to be non-adverse.

 

In conclusion, under the conditions of this study, test item related adverse effects were observed after 90 days of treatment in the high dose group; effects included signs of anaemia and erosion/ulcer in the stomach. There were test item related higher liver, epididymis and kidney organ weights at the high dose in both sexes. There were no adverse effects of test item in the mid and low dose groups of either sex. The NOAEL (no observed adverse effect level) of APD was considered to be 250 mg/kg bw/day.

Justification for classification or non-classification

The available data on the repeated dose toxicity 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.