2-(2,4-diaminophenoxy)ethanol dihydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline 408 and in compliance to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

EC Number:

266-357-1

EC Name:

2-(2,4-diaminophenoxy)ethanol dihydrochloride

Cas Number:

66422-95-5

Molecular formula:

C8H12N2O2.2ClH

IUPAC Name:

2-(2,4-diaminophenoxy)ethan-1-ol dihydrochloride

Details on test material:

Test item : 2,4-Diaminophenoxyethanol dihydrochloride
EC number : 266-357-1
Batch number : 0120022
Purity : >99.5%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source : Charles River Laboratories, France
-Age at study initiation : ~6 weeks
-Weight at study initiation : 195-233g for males ; 130-174g for females
-Assigned to test groups randomly : Yes
-Housing : Two rats of the same sex and group
-Diet : A04C pelleted maintenance diet, SAFE, France, ad libitum
-Water : Tap water, ad libitum
-Acclimation period : 6 days

ENVIRONMENTAL CONDITIONS
-Temperature : 22 +/-2 deg C
-Humidity : 30 to 70%
-Air changes (per hour) : ~12
-Photoperiod : 12 hour light/12 hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(purified, obtained by reverse osmosis)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
Before preparation, the vehicle was degassed for at least 15 minutes and then saturated with nitrogen gas and kept under nitrogen atmosphere for 15 minutes. The test substance was mixed with the vehicle in order to achieve the concentrations of 0.8, 4 and 20 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were prepared under a nitrogen atmosphere, weekly, according to the stability data obtained in CIT study number 26963 AHS and stored at +4 deg C prior to use. They were also protected from light and kept under nitrogen atmosphere until delivery.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, the suitability of the proposed preparation procedure was determined by the analysis of stability and concentration of the dosage forms, which were prepared using this procedure. During the treatment period, the concentration of samples taken from each dosage form (including the control) prepared for use in week 1, on day 17 and then in weeks 4, 8 and 13 was determined.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
4 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
20 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

140 animals total

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a previous inadequate 13-week oral toxicity study in which rats were given the test item at 56 mg/kg bw/day (Societe de Recherches Biologiques, 1978). In that study, the main effect observed was a slightly lower body weight gain. The high-dose for the present study was therefore set at 100 mg/kg bw/day as it was expected to produce sufficient toxic effects. The low and intermediate dose-levels were set at 4 and 20 mg/kg bw/day to assess the dose-relationship of any adverse effects.
- Rationale for animal assignment : During the acclimation period, the required number of animals (70 males and 70 females) were selected according to body weight and clinical condition and allocated to the groups (by sex), according to body weight, using a randomisation procedure, so that the average body weight of each group was similar.
- Assignment of animals : The animals were assigned into the following groups
Group 1 : vehicle control (principal - 10 males/10 females ; treatment-free - 6 males/6 females)
Group 2 : 4 mg/kg bw/day (principal - 10 males/10 females ; satellite - 6 males/6 females)
Group 3 : 20 mg/kg bw/day (principal - 10 males/10 females ; satellite - 6 males/6 females)
Group 4 : 100 mg/kg bw/day (principal - 10 males/10 females ; treatment-free - 6 males/6 females ; satellite - 6 males/6 females)

Examinations

Observations and examinations performed and frequency:

MORTALITY : Yes
- Time schedule : Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: The first day of dosing, weekly thereafter and at necropsy

FOOD CONSUMPTION :
- Time schedule : Recorded weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined before treatment started and at the end of the treatment period
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 1 and in week 13 at designated time-points

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 1 and in week 13 at designated time-points

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period

Sacrifice and pathology:

GROSS PATHOLOGY : Yes
-Time schedule for necropsy : On completion of the treatment or treatment-free periods, after at least 14 hours fasting, designated animals were anesthetised with carbon dioxide and then killed by exsanguination. After the last blood sampling, satellite animals were killed by carbon dioxide excess and discarded without necropsy.

HISTOPATHOLOGY : Yes

Other examinations:

ORGAN WEIGHTS : Yes

Statistics:

CITOX version D3 was used for the statistical analyses of body weight, food consumption, haematology, blood biochemistry, urinalysis and organ weight data. For motor activity evaluation, the following analyses were performed on rearing and horizontal movements : one way analysis of variance, in the case of significant differences in the results at the level of 5%, Dunnett test was implemented. These statistics were performed using specific software (SAS Enterprise Guide V2, SAS Institute Inc.).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY : No unscheduled deaths occurred during the study.

CLINICAL SIGNS : No relevant clinical signs were noted at 4 or 20 mg/kg bw/day. At 100 mg/kg bw/day, the main clinical sign recorded during the dosing period was ptyalism : it was observed in 15/16 males and 14/16 females for many weeks of dosing. This sign was also noted in satellite animals and was considered to be treatment related. It was not considered as an adverse effect as it is commonly encountered when a test item is administered by gavage. During the treatment free period, ptyalism was not observed in both males and females. The other clinical signs consisted of abnormal growth of teeth and cutaneous signs. As these signs were present either with a similar incidence in control and treated animals or sporadically in only a few animals, they were not attributed to treatment with the test item. Similar clinical signs were generally observed in satellite animals.

BODY WEIGHT AND WEIGHT GAIN : A similar mean body weight gain was recorded during the dosing period in controls and animals given 4 or 20 mg/kg bw/day. When compared to controls, slightly lower mean body weight and body weight gain were noted in males given 100 mg/kg bw/day during the whole dosing period. These changes which were statistically significant almost throughout the study were attributed to treatment with the test item. The small changes observed in mean body weight and body weight gain for females during the dosing period were minor, not statistically significant and not dose related. They were considered not to be treatment related. After a 4 week treatment free period, the mean body weight of males given 100 mg/kg bw/day was similar to controls.

FOOD CONSUMPTION : Similar mean food consumption was recorded, during the dosing period, in controls and animals given 4, 20 or 100 mg/kg bw/day.

OPHTHALMOSCOPIC EXAMINATION : There were no treatment related findings at the end of the treatment period.

HAEMATOLOGY : At the end of the treatment period, the minor differences observed in haematological parameters (monocytes, mean corpuscular haemoglobin concentration, activated partial thromboplastin time) between control and treated animals were not considered to be biologically significant as thet were of minimal to slight magnitude and isolated. All individual values remained within the range of control values and/or these changes were not recorded in both sexes.

CLINICAL CHEMISTRY : The differences observed in blood biochemical parameters between control and treated animals were of low magnitude, the contribution of only a few individuals, not observed in both sexes and the individual values were within CIT control data for most parameters. They were considered to be of no toxicological relevance.

URINALYSIS : There were no relevant changes in urinary parameters at 4 or 20 mg/kg bw/day. At the end of the treatment period, when compared to controls, the following findings were observed in the urine from animals given 100 mg/kg bw/day (presence of low to high bilirubin levels in all males and in 7/10 females, traces of nitrite in 8/10 males and 6/10 females, traces of glucose in 6/10 males and 6/10 females, marked colouration of urine (from yellow to yellow-brown) in both males and females).

NEUROBEHAVIOUR : There were no perturbations of the autonomic or physiological functions in any treated animals.The motor activity of both males and females measured over a 60 minute period was unaffected by treatment. When compared to controls, slightly higher numbers of rearing and horizontal movements were recorded for females given 100 mg/kg bw/day. As these changes were slight, not statistically significant and most probably due to the low control value, they were considered to be of no toxicological significance.

ORGAN WEIGHTS : No treatment related effects on organ weights were noted.

GROSS PATHOLOGY : Brownish discolouration was a feature in the thyroids of all animals given 100 mg/kg bw/day but not in the lower dose groups. This finding was also observed in animals at the end of the treatment free period.The other necropsy findings encountered were among those commonly recorded in the rat of this strain and age and none were considered to be toxicological importance.

HISTOPATHOLOGY: Brownish pigment was seen in the thyroids of all but one of the males and all females given 100 mg/kg bw/day. The one section in which it was not seen showed a level of artifact that could well have obscured the presence of pigment and consequently this section was disregarded from the count of this finding at this dose level. The pigment occurred as fine granules in the follicular epithelial cells and also in desquamated cells in the follicular lumen and occasionally in a parafollicular location. These pigment deposits were still observed in all animals given 100 mg/kg bw/day at the end of the treatment free period with similar incidence and severity to those noted at the end of the treatment period. These changes were not found in animals given lower dose levels (4 and 20 mg/kg bw/day). In the spleen, higher incidence and severity of hemosiderosis were noted in animals given 100 mg/kg bw/day. Hemosiderosis was still observed in all animals previously given 100 mg/kg bw/day at the end of the treatment free period with similar incidence and severity to those noted at the end of the treatment period. The severity of hemosiderosis was similar at lower dose levels and for controls.

Effect levels

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

urinalysis

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test substance 2,4-diaminophenoxyethanol dihydrochloride was administered daily by gavage to Sprague-Dawley rats at the dose level of 4, 20 or 100 mg/kg bw/day for 13 weeks. At 4 and 20 mg/kg bw/day the test item was well tolerated. A 100 mg/kg bw/day, ptyalism was observed in both males and females and lower body weight gains were noted for males. Presence of urinary bilirubin, nitrites, glucose and coloured urine in both males and females was observed at the end of the treatment period. After a 4 week treatment-free period, all the above-mentioned changes were no longer noted. Deposition of brownish pigment in the thyroids and an augmented degree of spleen hemosiderosis were also observed for most animals given 100 mg/kg bw/day on completion of treatment and treatment-free periods. Under the experimental conditions of the study, the NOAEL is established to be 20 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item 2,4 -diaminophenoxyethanol dihydrochloride following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4 -week treatment free period in order to evaluate the reversibility of any findings.

A total of 140 Sprague-Dawley rats (70 males/70 females) were allocated to three treatment groups and one control group. Each group was composed of 10 males and 10 females. Recover animals (6 males and six females) were added to the control and high dose groups for a 4 -week treatment free period. The test item was administered daily by gavage as a solution in the vehicle (purified water) at the dose level of 4, 20 or 100 mg/kg bw/day for 13 weeks. Control animals received the vehicle alone under the same experimental conditions. The animals were checked daily for mortality and clinical signs. detailed clinical observations were carried out weekly and a functional observation battery was conducted at the end of the treatment period. Body weight and food consumption were recorded once a week throughout the study. Ophthalmological examinations were performed before and at the end of the treatment period. Haematological and blood biochemical investigations and urinalysis were performed at the end of the treatment period. On completion of the treatment or treatment-free periods, the animals were sacrificed and submitted to a full macroscopic examination. Designated organs were weighed and specified tissues preserved. A microscopic examination was performed on selected tissues from animals in the control and high dose groups and on macroscopic lesions from all animals killed on completion of the treatment period. The thyroids and spleen were submitted to microscopic examination for all low and intermediate dose animals at the end of the dosing period as well as for high dose animals at the end of the treatment free period.

No unscheduled deaths occurred during the study. Ptyalism, noted among almost all the animals given 100 mg/kg bw/day, was not considered as an adverse effect as it is commonly encountered when a test item is administered by gavage. There were no perturbations of the autonomic or physiological functions in any treated group. A slightly lower mean body weight gain was noted in males given 100 mg/kg bw/day during the whole dosing period and was most of the time statistically significant. Mean male body weight returned to control values during the treatment free period. No treatment related effect was noted on female body weight gain. No treatment related effects were noted on food consumption for either males or females and there were no ophthalmological treatment related findings at the end of the treatment period. No treatment related changes were noted for any haematological or blood biochemical parameters. At the end of the treatment period, the following treatment related differences from controls were observed in urinary parameters at 100 mg/kg bw/day : presence of low to high bilirubin levels in all males and in 7/10 females, traces of nitrite in 8/10 males and 6/10 females, traces of glucose in 6/10 males and 6/10 females, marked colouration of urine (from yellow to yellow-brown) in both males and females. All these changes were attributed to treatment and were no longer present on completion of the treatment free period. No treatment related effects on organ weights were noted. Brownish colouration of the thyroids was observed in all males and females given 100 mg/kg bw/day and was associated at microscopic examination with brownish pigment deposits in the thyroids. Spleen hemosiderosis was also observed for all animals given 100 mg/kg bw/day. Both these microscopic changes on thyroids and spleen were still observed at the end of the recovery period.

The test substance 2,4-diaminophenoxyethanol dihydrochloride was administered daily by gavage to Sprague-Dawley rats at the dose level of 4, 20 or 100 mg/kg bw/day for 13 weeks. At 4 and 20 mg/kg bw/day the test item was well tolerated. A 100 mg/kg bw/day, ptyalism was observed in both males and females and lower body weight gains were noted for males. Presence of urinary bilirubin, nitrites, glucose and coloured urine in both males and females was observed at the end of the treatment period. After a 4 week treatment-free period, all the above-mentioned changes were no longer noted. Deposition of brownish pigment in the thyroids and an augmented degree of spleen hemosiderosis were also observed for most animals given 100 mg/kg bw/day on completion of treatment and treatment-free periods. Under the experimental conditions of the study, the NOAEL is established to be 20 mg/kg bw/day.