Registration Dossier

Administrative data

Description of key information

A sub-chronic toxicity study (according to OECD 408) has been be conducted using the source substance APD. The results have been used for read-across to TRIS AMINO based on an analogue approach as permitted according to Regulation (EC) 1907/2006, Annex XI. The NOAEL was considered to be 250 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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 jusitification for read-across has been attached in section 13 of this IUCLID.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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 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:
organ weights and organ / body weight ratios
gross pathology
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:
liver
kidney
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:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
hepatobiliary

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

Additional information

There are no data available on the sub-chronic toxicity of 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS AMINO). In order to adapt the standard information requirements, a GLP-compliant sub-chronic toxicity study in the rat via the oral route following OECD 408 has been performed, using the source substance 2-amino-1,3-propanediol (APD). The potential analogues for the target substance TRIS AMINO are other 2-amino-1,3-propanediols. Members of the aminopropanediol category: 2-amino-2-ethyl-1,3-propanediol (AEPD), 2-amino-2-methyl-1,3-propanediol (AMPD) and 2-amino-1,3-propanediol (APD). Based on the available experimental data on several 2-amino-1,3-propanediols and their similar physico-chemical characteristics, the substances are expected to show comparable toxicokinetic characteristics. TRIS AMINO administered via the oral or dermal route will only be absorbed in small amounts (Brinkman et al., 1960; Noel-Hudson, 1993). The available acute toxicity studies show low toxic potential via the oral and dermal route for all the source substances, supporting the theory that all the aminopropanediols will be absorbed in limited amounts and have low systemic bioavailability. TRIS AMINO is not metabolised in-vivo (Brasch and Iven, 1981).

 

The modelling of potential metabolites using the OECD QSAR toolbox v.2.0 (2010) did not predict relevant metabolites of TRIS AMINO, as was shown by experimental data, or of any of the 2-amino-1,3-propanediols. Therefore, no metabolism by cytochrome P450 enzymes in-vivo is expected. TRIS AMINO is rapidly eliminated via the kidneys (Brasch and Iven, 1981; Brasch et al., 1982). As all the source substances are polar, highly water soluble and have a molecular weight below 500, their elimination is also expected to occur mainly via the kidneys. Available studies via the oral, dermal or intraperitoneal route on these substances caused no systemic toxicity. 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 respective test substances at the site of contact. The Cramer classification (related mainly to the oral route) also indicates a low toxicological concern for TRIS AMINO and the 2-amino-1,3-propanediols. Thus, both TRIS AMINO and 2-amino-1,3-propanediols are of low concern with regard to systemic toxicity. Due to the structural similarity and the similar toxicological properties between TRIS AMINO and the 2-amino-1,3-propanediols, read-across between these substances using an analogue approach is justified as outlined in Regulation (EC) 1907/2006, Annex XI, also to avoid tests for animal welfare reasons.

 

Repeated dose toxicity: oral  

A reproduction/developmental toxicity screening test was performed with 2-amino-2-(hydroxymethyl)-1,3-propanediol (trometamol), according to OECD 421 (Ellis-Hutchings, 2012). Rats (12/sex/dose) were administered 100, 300 and 1000 mg/kg bw/day of the test substance, adjusted to pH 9, by gavage. Males were exposed for 29 days and females for up to 54 days (from 14 days before mating until day 4 of lactation). There was no mortality during the study period and no treatment-related clinical signs or body weight changes were observed. There were no effects on organ weights. During the gross pathology, a thickened limiting ridge of the stomach was observed in all the males and females in the high-dose groups. Effects on the limiting ridge were also seen in the microscopic examination, as very slight to slight hyperplasia of the stratified squamous epithelium in all animals in the high-dose group. A few cases were also noted in the control or low- and mid-dose groups. A clear treatment-related effect was seen only in the cases classified as slight; with an increasing number of cases by increasing dose. The damage to the limiting ridge is most probably caused by local irritation of the test substance (adjusted to pH 9), which was administered in a relatively large volume. As humans do not have a limiting ridge and forestomach, the effect is considered to have limited toxicological relevance to humans. There were no treatment-related histopathological findings in the reproductive organs.

Therefore, the NOAEL systemic in this OECD 421 study is considered to be > 1000 mg/kg bw/day for males and females in this study. Based on the local irritation effects the NOAEL local is set at 100 mg/kg bw/day.

 

Several publications on oral repeated dose toxicity studies performed with trometamol prior to the OECD guidelines are available. Darby and Anderson (1966) performed two studies on rats, recording mortality, clinical signs and performing gross pathology: In the 15-day study, the NOAEL was ≥ 2500 mg/kg bw/day, as no effects were seen up to and including the highest dose level. In the 31-day study, diarrhoea was observed at the highest dose level. Although this is a treatment-related effect, it is not likely to be an irreversible adverse effect. Therefore, the NOAEL is considered to be > 4000 mg/kg bw/day. In a subacute study, 10 rats/dose level were administered 1 or 2% (equivalent to approximately 1000 and 2000 mg/kg bw/day) of the test substance in the diet for 35 days (Giroux and Beaulaton, 1961). The mortality, clinical signs and body weight were recorded. At necropsy, gross pathology was performed and the stomachs of 5 animals from each group were subject to histopathological examination. A satellite group of 5 animals in each dose group was kept an additional 15 days. No effects were observed up to and including the highest dose level and there was no difference between the main treatment groups and satellite treatment groups.

 

In a sub-acute study dogs were administered 250, 1000 and 4000 mg/kg bw/day for 30 days (Darby and Anderson, 1966). Mortality and incidence of clinical signs was registered and gross pathology was performed on all animals, 12 in total. Vomiting and loose stools were noted occasionally in dogs administered 1000 mg/kg bw/day, and frequently in dogs administered 4000 mg/kg bw/day. These effects are similar to those observed in the rats and are considered to be treatment-related, though not irreversible adverse effects. The NOAEL is considered to be > 4000 mg/kg bw/day, the highest dose level tested. 

 

A sub-chronic toxicity study (according to OECD 408) has been be conducted using the source substance APD. The results have been used for read-across to TRIS AMINO based on an analogue approach as permitted according to Regulation (EC) 1907/2006, Annex XI. 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 was considered to be 250 mg/kg bw/day.

 

Repeated dose toxicity: dermal

In a study report containing limited data, the potential dermal toxicity of trometamol in rabbits was evaluated (Machle et al., 1940). An unknown amount of the test substance was applied to the shaved skin for 4 hours without cover for 5 consecutive days. No local skin effects or clinical signs were observed and the body weight was not affected.

 

Repeated dose toxicity: other routes

Several intravenous and intraperitoneal repeated dose toxicity studies performed with trometamol prior to the OECD guidelines are available. A study was performed in rabbits, in which 4 rabbits/dose/sex received an intravenous injection of 500 mg/kg bw/day trometamol, 5 days/week for 28 days (Thompson, 1965). The animals were observed for up to 20 days after the exposure ended. Local inflammatory lesions were observed in 7/8 rabbits around the injection site on the ear. 2/4 rabbits in the recovery group had chronic interstitial nephritis, while 1/4 had peracute toxic nephrosis. These effects were considered to be treatment-related and caused by the alkalinity of the test substance. The LOAEL i.v. was considered to be 500 mg/kg bw/day. In a study by Thompson (1965), 3-6 rats/group received daily doses of 0.3 M trometamol by intravenous injection (equivalent to 500 mg/kg bw/day) or intraperitoneal injection (equivalent to 1500 mg/kg bw/day) for 20 days, or received 10 days treatment per exposure route. Local effects observed at the i.v. and i.p. injection sites of approximately half the animals were attributed to the alkaline effect of the test substance. Peracute toxic nephrosis was observed in 5/6 rats in group 1 (i.v.); 6/6 in group 2 (i.p.); and 2/6 in group 3 (10 days i.v. and 10 days i.p.). Following a 20-day recovery period, only 5/6 rats in the satellite group 2 were affected. The LOAEL i.v. was considered to be 500 mg/mg bw/day, while the LOAEL i.p. was considered to be 1500 mg/kg bw/day.

 

Several additional studies were performed, in which very limited data was reported (Darby and Anderson, 1966; Mulinos, 1961; Roberts and Linn, 1961). The test substance was administered i.v. to rabbits, mice and dogs, or i.p. to dogs in doses from approximately 186 to 3000 mg/kg bw/day. The overall trend in these studies is that high doses (from approximately 1500 mg/kg bw/day) may cause adverse effects (convulsions, vomiting in dogs) or mortality in rabbits and dogs, while lower doses do not affect the animals. The observed effects were generally attributed by the authors to the alkalinity of the test substance.

 

Justification for classification or non-classification

The available data on the repeated dose toxicity of the test substance and other members of the category of aminopropanediols 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.