2-aminoethanol

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the studies reported herein was to investigate the potential role of choline antagonism in the aetiology of Ethanolamine (EA; 2-aminoethanol; CAS RN 141-43-5)-induced implantation loss. The work was conducted in two phases: the first to determine if there was a critical period of sensitivity during the pre- and peri-implantation period; the second to evaluate the impact of choline co-administration on implantation success following treatment with EA.

GLP compliance:

yes

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

2-aminoethanol

EC Number:

205-483-3

EC Name:

2-aminoethanol

Cas Number:

141-43-5

Molecular formula:

C2H7NO

IUPAC Name:

2-aminoethanol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Housing: housed singly in Makrolon type M III cages, with dust-free wood chipbedding and suitable environmental enrichment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24
- Humidity (%): 30–70
- Photoperiod: 12/12

Administration / exposure

Route of administration:

other: drinking water (1st Experiment) and diet (2nd experiment)

Details on exposure:

1st Experiment:
Groups of twelve time-mated pregnant female rats were administered either EA-HCl (1000 mg/kg bw/day; 10.25 mmol/kg bw/day) or IPEA (100 mg/kg bw/day; 0.97 mmol/kg bw/day) in distilled water, once daily by gastric intubation on GD 1–3, GD 4–5, or GD 6–7. A control group was administered distilled water through-out the period GD 1–7. The IPEA solution was adjusted to pH 7.0 by the addition of hydrochloric acid prior to administration. Blood was collected by retroorbital venous puncture under isoflurane anaesthesia for standard haematological and clinical chemistry analyses. Food consumption and body weight were recorded throughout gestation, and daily cage-side checks were made for clinical signs of toxicity or morbidity. On GD 20, all animals were anaesthetised with isoflurane and sacrificed by cervical dislocation. After grosspathological examination, the ovaries were removed and the corpora lutea were counted, and the uterus was removed and weighed. The uterus was then opened and evaluated for the number of implantations which were differentiated according to live and dead foetuses, and early or late resorptions. Early resorptions in animals that did not appear to be pregnant, or which had single-horn pregnancy, were confirmed by ammonium sulphide staining. Foetuses were sacrificed by subcutaneous injection of sodium pentobarbital.

2nd Experiment:
After a seven-day acclimatisation period, groups of twelve non-pregnant female rats were given standard diet or diet supplemented with choline chloride to achieve choline intakes of approximately 1.2–1.5 and 6.2–7.5 mmol/kg bw/day respectively. Subsets of these groups were administered EA.HCl (1511 mg/kg bw/day;15.49 mmol/kg bw/day) in distilled water adjusted to pH 7.0 with aqueous sodium hydroxide, once daily by gastric intubation; EA.HCl in the diet at an inclusion level to
achieve an equivalent delivered daily dose; or distilled water by oral gastric intubation. A higher dose of EA was selected compared to that used in Experiment 1, based on the OECD limit dose of 1000 mg/kg/day as EAfree base. Therefore, there were six experimental groups in a 2 × 3 block design: untreated control (group 0); choline-supplemented diet (group 1); EA administered by gastric intubation (group 2); EA administered by gastric intubation with choline-supplemented diet (group 3); EA-su
pplemented diet (group 4); and diet supplemented with both EA and choline (group 5). After fourteen days of treatment each female was paired overnight with an untreated male in the male’s home cage. In cases where females were treated via the diet, the males’ diet was replaced by that of the females. The pairing procedure continued until evidence of copulation, either vaginal plug or sperm in the vaginal smear. This day was denoted GD 0, after which the females remained in their owncages and were treated as before until GD 8. Blood was collected approximately 1–1.5 hours after the final dose administration, by retroorbital venous puncture under isoflurane anaesthesia, for standard haematological and clinical chemistry analyses as well as the measurement of EA concentration in the plasma. All animals were then given standard untreated diet from GD 9 onwards and gavage treatment was curtailed. Food consumption and bodyweight were recorded throughout gestation, and daily cage-side checks were made for clinical signs of toxicity or morbidity. The experiment was terminated on GD 20 in the same manner as the first.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of EA.HCl and choline chloride in the die twere determined by HPLC/MS, monitoring the protonated [M + H]+ions formed by heated electrospray ionisation. A sample of the treated diet was accurately weighed and suspended in 0.1% aqueous formic acid by shaking for one hour. An aliquot was then diluted further with 0.1% aqueous formic acid and filtered through a Millex-GV 0.45 μm filter prior to direct injection (10 μL) onto the column (Primesep 200, 250 × 3.2 mm, 5 μm). Separation was by means of gradient elution between water and acetonitrile, each containing 0.1% formic acid, at a constant flow rate of 0.5 mL/min. The concentration of EA (as free base) in rat serum was also determined by HPLC/MS. An aliquot (20 μL) of serum was mixed with methanol (980 μL), centrifuged (13000 rpm, 5 min), and an aliquot (10 μL) of the supernatant was applied directly to the column. The mobile phase consisted of 80% acetonitrile and 20%aqueous ammonium formate (50 mM, pH 3.7) at a flow rate of0.5 mL/min. Again, the protonated [M + H]+ions were monitored following heated electrospray ionisation in positive mode. The working limit of quantitation was approximately 50 ng/mL; in comparison, the concentration of EA in serum from an untreated rat was 445 ng/mL.

Duration of treatment / exposure:

see "Details on exposure"

Frequency of treatment:

see "Details on exposure"

Duration of test:

see "Details on exposure"

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

EA treatment in 1st Experiment

Doses / concentrations 2

Dose / conc.:

1 511 mg/kg bw/day (nominal)

Remarks:

EA treatment in 2nd Experiment

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

see "Details on exposure"

Statistics:

If Bartlett’s test for homoscedasticity was not significant, fol-lowing transformation if necessary, continuous data were analysedby unpaired t-test or by ANOVA followed by either Dunnett’stest (experiment 1) or the Tukey-Kramer test (experiment 2); the central tendency is presented as the mean, with dispersion as standard error. Proportional data were analysed by Chi2 and Fisher’s exact tests. Count data, and continuous data for which parametric analysis was not appropriate, were analysed by unpaired U-test or by Kruskal-Wallis ANOVA followed by either Steel’s test (experiment
1) or the Dwass-Steel-Critchlow-Fligner test (experiment 2); the central tendency is presented as the median, with dispersion represented by first and third quartiles. Multiple pair-wise comparisons were correctedwhere necessary. Growth (that isrepeated-measures of body weight) was evaluated with multilevel mixed-effects linear regression. The effects of EA and IPEA administered during the pre- and peri-implantation period (experiment 1) and the effect of EA administered by gastric intubation or diet(experiment 2; comparing groups 0, 2, and 4, and comparing groups 1, 3, and 5) were tested by parametric or Kruskal-Wallis ANOVA followed by appropriate post hoc tests. The interaction between choline and EA (experiment 2; comparing group 3 with group 2, and group 5 with group 4) was tested by unpaired t- or U tests. Preimplantation loss per litter was calculated as the difference between counts of corpora lutea and total implantation sites; post-implantation loss per litter was calculated as the difference between total implantation sites and live foetuses.

Results and discussion

Effect levels

Key result

Remarks on result:

not measured/tested

Observed effects

1st Experiment:
Treatment with EA.HCl (1000 mg/kg bw/day; 10.25 mmol/kg bw/day) or with IPEA (100 mg/kg bw/day; 0.97 mmol/kg bw/day) during GD 6-7 had no consistent effect upon food intakeduring the period of treatment or thereafter. Treatmentwith IPEA during GD 1-3 or 4-5, however, resulted in substantially reduced food intake during the final trimester. EA caused aminor reduction in body weight compared to the concurrent control group only when administered during the periods GD 1-3 and GD 4-5; body weight gain was significantly reduced from GD 13, but mean body weight values were within 5% of the control group value and only attained statistical significance from GD 19. IPEA caused a substantial reduction in body weight when administer
ed during the same periods. In both cases, administration during GD 6-7 had little or no effect on body weight gain. There were no dead foetuses resulting from the administration of either EA or IPEA. Furthermore, treatment with EA had no effect upon the number of implantation sites, early resorptions, or live foetuses. Total litter resorption in one dam treated during GD 4-5, which involved a single implant, is considered to be an isolated event and unrelated to treatment. Treatment with IPEA resulted in a strong response, most notably in terms of total pre-implantation loss among animals treated during GD 1–3; of the twelve animals that were mated, eleven were found to be not pregnant, as opposed to one of twelve in the control group. When treatment was delayed to the period GD 4-5, the response was manifested as an early loss of implantations. Treatment later in the peri-implantation period had no effect upon the number of either implants or live foetuses. Total litter resorption of five and eleven implants in one dam from the GD 1-3 and GD 6-7 treatment groups respectively may reflect the variability in actual timing of conception compared to the nominal assignment.
2nd Experiment:
Treatment with EA (1511 mg/kg bw/day, 15.49 mmol/kg bw/day) by gastric intubation had no effect upon food consumption and only marginal effect on body weight gain towards the end of gestation. Dietary administration of EA resulted in slightly reduced food consumption, slight reduction in body weight gain during the latter part of the administration period, and a slight reduction on body weight compared to control during the latter part of gestation. The overall intake of EA by both routes was
closely matched. The inclusion of choline in the diet resulted in an approximately fivefold increase in choline intake, and a reduction in food consumption which was reversed on cessation of treatment at GD 8, the latter of which was reflected in reduced body weight increase. Nevertheless, body weights of all EA-treated groups were within 10% of the respective control group values. Time to successful mating was unaffected by treatment. There were no dead foetuses and, with the exception of one implant in group 1, no late resorptions .The administration of EA resulted in an increase in pre- and postimplantation losses, although this only reached statistical significance for post-implantation loss when EA was administered in the diet. The co-administration of choline ameliorated the pre- and postimplantation loss induced by EA when administered both by gastric intubation and via the diet. Although this was only statistically significant for the induction of pre-implantation loss by dietary EA (p < 0.05), the induction of post-implantation loss by EA dosed as a bolus was reduced by borderline statistical significance (p = 0.054). Gavage administration resulted in a higher plasma concentration than dietary administration. The co-administration of choline significantly reduced EA concentrations in both cases (p < 0.05).