Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached (in chapter 13 of IUCLID) document with the justification for the category/read-across approach.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): 2-amino-2-ethyl-1 ,3-propanediol
- Physical state: Transparent, yellow, viscous liquid
- Analytical purity: 99.4%
- Stability under test conditions: The stability of the test substance was tested by diluting it to 30 and 200 mg/mL in water and storing the solutions in light-proof bottles under refrigeration, in the dark, for 6 days. The test substance was diluted with water for injection to 25, 50, and 100 mg/mL solutions. 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). The solutions were confirmed to be stable under these conditions.
- Storage condition of test material: Cool and dark conditions

Specific details on test material used for the study:
Test Substance name: 1,3-Propanediol, 2-amino-2-ethyl
CAS number: 115-70-8
Purity: 99.4%
Properties: transparent yellow, viscous liquid, no pollution or visible contamination observed.
Stability:post animal testing completion, the result analyzed by BOZO Research Center results showed that the test substance was stable during the animal test period.
Storage method: cool dark location (actual measured value: 2 to 8°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley Crj:CD9SD)IGS SPF
Sex:
male/female
Details on test animals 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

Administration / exposure

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 dosing solutions were verified twice using GC, before the first administration and in the final week of the administration.
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
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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
Positive control:
not applicable

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No effects on clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in body weight were observed between the control groups and the treatment groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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, 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.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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 findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant decrease in the number of rearings (standing on rear limbs) 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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested

Target system / organ toxicity

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:
no

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, it was determined that the NOAEL for repeated dose toxicity was 500 mg/kg/day for males and 1000 mg/kg/day for females.
Executive summary:

In order to investigate the toxicity of repeated administration and toxicity on reproduction, 2-amino-2-ethyl-1,3-propanediol (AEPD) was administered to Sprangue-Dawley rats at doses of 0 (control group), 250, 500 and 1000 mg/kg bw. Males received the substance during 14 days before mating and during the mating period till the day before autopsy (42 days of administration). Females received the substance during 14 days before mating and during the mating period and during the gestation period till the 4th day of nursing (42 to 48 days of administration). For the control 0 and 1000 mg/kg group, a recovery period of 14 days was set and reversibility of potenial effects was investigated.

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. Based on the results of the study, it was determined that the NOAEL for repeated dose toxicity was 500 mg/kg/day for males and 1000 mg/kg/day for females.