2-(2,4-diaminophenoxy)ethanol dihydrochloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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 414 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 414 (Prenatal Developmental Toxicity Study)

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source : Charles River Laboratories, France
-Age at study initiation : 10-11 weeks
-Weight at study initiation : 204-315g
-Assigned to test groups randomly : According to a stratification procedure based on body weight to ensure a comparatively similar mean body weight among the groups
-Housing : Individually
-Diet : A04C pelleted maintenance diet, SAFE, France, ad libitum
-Water : Tap water, ad libitum
-Acclimation period : 7 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 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 25 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. These analyses were performed in CIT study number 26963 AHS at concentrations spanning those used in the present study. The concentration of samples taken from each control and test item dosage form prepared for use on the first and the last day of the dosing period was determined according to the analytical method previously validated in CIT study number 26963 AHS.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:2
- Length of cohabitation: The females in proestrus were placed overnight in the home cage of singly housed males of the same strain. Each morning following mating, rats with spermatozoa found in a vaginal smear or a sperm plug in situ were considered as pregnant animals.
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: See above

Duration of treatment / exposure:

Animals were treated on days 6 to 19 of gestation

Frequency of treatment:

Animals were treated once daily

Duration of test:

Animals were sacrificed on day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day

No. of animals per sex per dose:

Number of mated females per dose : 24

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-level selection : The dose levels were selected in agreement with the sponsor based on the results of a preliminary range-finding study in embryofetal development. The high dose level was set at 125 mg/kg bw/daybased on the results of a preliminary study performed at 100, 150 and 200 mg/kg (CIT study number 26997 RSR). In that study, dose related maternal toxicity was observed at all dose levels and consisted of decreased body weight gain (-18%, -38% and -35% at 100, 150 and 200 mg/kg respectively) and food intake. Marked clinical signs were also observed at 200 mg/kg bw/day and resulted in the death of one female. These maternal effects were associated with a dose-related decrease in fetal weight at all dose levels. Based on these results, it was anticipated that the dose level of 125 mg/kg would be associated with sufficient maternal toxicity and was therefore selected as the high dose level for the present study. Low and intermediate dose levels were set at 4 and 20 mg/kg bw/day to assess the dose relationship of the toxic effects.

Examinations

Maternal examinations:

Morbidity and mortality : Each animal was checked for mortality or signs of morbidity at least twice a day during the treatment period and at least once a day on other days.

Climical observations : From arrival, the animals were observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time for the recording of clinical signs.

Body weight : The body weight of each female was recorded on days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption : The quantity of food consumed by each female was recorded for the following intervals : days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20 post coitum.

Ovaries and uterine content:

The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus). The ovaries and uterus were examined to determine :
- number of corpora lutea
- number and distribution of dead and live fetuses
- number and distribution of early and late resorptions
- number and distribution of uterine scars
- number and distribution of implantation sites
A gross evaluation of placentas was also undertaken.

Fetal examinations:

These examinations were carried out for all the litters.

Body weight : The body weight of each live fetus was recorded.

External examination : Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

Soft tissue examination : A detailed soft tissue examination was performed according to a serial sectioning technique which included the observation of all the organs and structures of the head, neck, thorax and abdomen.

Skeletal examination : A detailed examination of the skeleton was performed including the observation of all the bone structures of the head, spine, rib cage, pelvis and limbs.

Sex of fetuses : The sex of each live fetus was determined.

Statistics:

Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 125 mg/kg bw/day, excessive salivation was transiently observed in 11/24 females. Body weight gain was statistically significantly reduced throughout the dosing period resulting in an overall reduction of 24% during the dosing period (days 6 to 20 post coitum), when compared to controls (p<0.001). The group mean food consumption was statistically significantly reduced throughout the dosing period, when compared to controls. At 4 and 20 mg/kg bw/day, there was no maternal toxicity.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity (reduced body weight gain, reduced mean feed consumption)

2

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There was a statistically significant reduction in the mean fetal weight (-7%, p<0.001) associated with a statistically significantly increased incidence of fetuses showing incomplete ossification of thoracic vertebra centrum or supernumeracy short 14th rib. At 4 and 20 mg/kgbw/day, there was no effects on the embryo-fetal development.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal variation, incomplete ossification

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

MATERNAL DATA

Pregnancy status :

Dose level/mg/kg bw/day	0	4	20	125
Mated females	24	24	24	24
Non-pregnant females	0	2	0	0
Pregnant females subjected to fetal examination	24	22	24	24

Mortality : No premature deaths occurred during the study.

Clinical signs : Nine females receiving 125 mg/kg bw/day were observed with excessive salivation from ~ day 15 of gestation until the end of treatment on day 19 of gestation. Two other females in this group had excessive salivation on day 18 or day 19 of gestation only. This finding was considered to be treatment related. No clinical signs were observed in females receiving 4 or 20 mg/kg bw/day.

Body weight : At 125 mg/kg bw/day, the body weight gain was statistically significantly lower throughout the dosing period, resulting in an overall reduction of -24% in weight gain from day 6 to 20, when compared to the controls. This resulted in significantly reduced body weights from day 12 of gestation onwards. When the net weight change was calculated by correcting for gravid uterus weight, the group mean body weight change was 47% of the control group mean. The mean carcass weight was significantly lower at 125 mg/kg bw/day when compared to controls. The treatment had no effect on body weights or body weight gains for the groups receiving 4 or 20 mg/kg bw/day.

Food consumption : Group mean food consumption was statistically significantly lower for the group receiving 125 mg/kg bw/day during the whole dosing period (between -11% and -16%) when compared with controls. There was no effect of treatment on group mean food consumption for the groups receiving 4 or 20 mg/kg bw/day.

Necropsy findings : No findings recorded at maternal necropsy were considered to be related to treatment.

Gravid uterus weight : The slight differences noted for gravid uterus weight in the treated groups, when compared to controls, were minor, not statistically significant, not dose-related and considered to reflect variations of the number of live fetuses in addition to fetal body weight.

Litter data : There were no effects on the group mean numbers of implantations or live fetuses, on the extent of pre- or post-implantation losses, or on the fetal sex ratio in any group. There were no dead fetuses except one at 125 mg/kg bw/day. The group mean fetal weight was unaffected by treatment at 4 and 20 mg/kg bw/day while it was significantly reduced at 125 mg/kg bw/day when compared to controls.

EXTERNAL OBSERVATIONS

There were no external malformations or variations observed in the groups. The only abnormalities were noted in one fetus from the control group which presented anasarca, ectrodactyly, bilateral short digits, gastroschisis and bent tail.

SOFT TISSUE OBSERVATIONS

Soft tissue malformation : No soft tissue malformations were observed in any group.

Soft tissue variation : No variations were considered to be treatment related as they were not recorded in the high dose group. The variations (presence of contents in stomach, dilated ureter and renal pelvis) were mainly observed in the control and the low and intermediate dose groups with most of the time a higher incidence in controls.

SKELETAL OBSERVATIONS

Skeletal malformations : There were four fetuses in the control group and a single etus in the 125 mg/kg bw/day group showing skeletal malformations. These findings were considered to be of spontaneous origin and not treatment related.

Skeletal variations : At 125 mg/kg bw/day the following statistical differences were recorded :

- a statistically significant increase in the mean percentage of fetuses with incomplete ossification of the centrum of the thoracic vertebrae

- a statistically significant higher number of fetuses with incomplete ossification of the 5th sternebra (not considered treatment related as the mean incidence of non ossification of this bone was decreased (fetal incidence : 21%, 11%, 4% at 0, 20 and 125 mg/kg bw/day respectively)

- the mean number of fetuses with a short supernumerary 14th rib was markedly higher. This finding when expressed as a mean percentage of affected fetuses per litter was outside the background data range and was therefore considered to be related to the treatment.

Applicant's summary and conclusion

Conclusions:

The test item, 2,4-diaminophenoxyethanol dihydrochloride, administered daily by gavage to pregnant female Sprague-Dawley rats from days 6 to 19 of gestation was well tolerated at 4 and 20 mg/kg bw/day. At 125 mg/kg bw/day, 11/24 females presented excessive salivation. The maternal body weight gain and food consumption were significantly lower throughout the dosing period. A slightly significantly lower fetal weight was observed at this dose-level, associated with a slightly higher number of fetuses with incomplete ossification of thoracic vertebra centrum and an increased incidence of short supernumerary 14th ribs. Under these experimental conditions, the NOEL for maternal toxicity and embryofetal development are 20 mg/kg bw/day.

Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item 2,4 -diaminophenoxyethanol dihydrochloride on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to scheduled hysterectomy (day 6 to day 19 of gestation inclusive).

Three groups of 24 mated female rats of the Sprague-Dawley strain received the test item at 4, 20 or 125 mg/kg bw/day. Another group of 24 females acted as a control group and received the vehicle, purified water, under the same experimental conditions. Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals. On day 20 of gestation, the dams were sacrificed and subjected to a macroscopic examination. The gravid uterus was weighed and then the fetuses were removed. The numbers of corpora lutea, implantation sites, early and late resorptions and dead and live fetuses were recorded. The fetuses were weighed, sexed and subjected to external, visceral or skeletal examinations.

At 125 mg/kg bw/day, excessive salivation was transiently observed in 11/24 females. Body weight gain was statistically significantly reduced throughout the dosing period resulting in an overall reduction of 24% during the dosing period (days 6 to 20 post coitum), when compared to controls. The group mean food consumption was statistically significantly reduced throughout the dosing period when compared to controls. There was a statistically significant reduction in the mean fetal weight (-7%,) associated with a statistically significantly increased incidence of fetuses showing incomplete ossification of thoracic vertebra centrum or supernumerary short 14th rib. At 4 and 20 mg/kg bw/day, there was no maternal toxicity and no effects on the embryo-fetal development.

The test item, 2,4-diaminophenoxyethanol dihydrochloride, administered daily by gavage to pregnant female Sprague-Dawley rats from days 6 to 19 of gestation was well tolerated at 4 and 20 mg/kg bw/day. At 125 mg/kg bw/day, 11/24 females presented excessive salivation. The maternal body weight gain and food consumption were significantly lower throughout the dosing period. A slightly significantly lower fetal weight was observed at this dose-level, associated with a slightly higher number of fetuses with incomplete ossification of thoracic vertebra centrum and an increased incidence of short supernumerary 14th ribs. Under these experimental conditions, the NOEL for maternal toxicity and embryofetal development are 20 mg/kg bw/day.