2-amino-2-ethylpropanediol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

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

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

ovary weights were not recorded

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-2-ethyl-1,3-propanediol

IUPAC Name:

2-amino-2-ethyl-1,3-propanediol

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

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crj:CD9SD)IGS SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Japan Co., Japan
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: (P) 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Females were housed individually in plastic Ekon cages with flooring (white flakes; Charles River Japan Co.) from day 17 of pregnancy to day 4 of lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the solutions used in the administration were measured twice by GC, before the first administration and in the final week of the administration, and were found to have suitable concentrations.

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

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

All of the females confirmed to have mated were allowed to deliver naturally and any difficulties delivering were noted. The completion of delivery was checked twice a day (a.m. and p.m.) up to from day 21 of pregnancy to the afternoon of day 22 of pregnancy and the pregnancy period was calculated in the unit of 0.5 days. If the deliveries were completed at 5:00 p.m., that day was taken as day 0 of nursing; the pregnancy period (days) (day 0 of nursing–day 0 of pregnancy) and the delivery rate (%) [(number of females delivering surviving pups)/(number of pregnant females) x 100] were calculated.
The incidence of muricide of the delivered pups and the treatment of the placentas and amnia were observed and the day of the delivery was noted as day 0 of lactation. The surviving pups were nursed up to lactation day 4 and the succes rate was observed, using the picking up of the pups, taking care of the nests, and the nursing as parameters.

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg
Basis:
other: nominal dose

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 groups. In addition, a satellite group of 5 animals per sex was included in the control and 1000 mg/kg bw 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 for 2 weeks, no effects of the test substance were observed. 1000 mg/kg bw 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 as the highest dose and 500 and 250 mg/kg bw 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 groups.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:

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 AND COMPOUND INTAKE (if feeding study):
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

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 apolipaseprotein (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.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily after the observation of the estrous cycle up to the day of group allotment and continuing until the confirmation of mating. The smears were examined microscopically. The vaginal smear images were classified into the pre-estrous, estrous, post-estrous, and estrous dormancy periods during the premating administration period; the number of days until the next estrous cycle was investigated on the basis of the number of appearances of the estrous cycle parameters and the number of estrous cylces was determined. Only animals with a normal estrous cycle were used in the study.

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
testis weight, epididymis weight.

Litter observations:

PARAMETERS EXAMINED
The numbers of surviving pups and stillborn pups were counted on the day of delivery. Abnormalities in the external appearances of the pups were observed, the numbers of each sex were counted, and their body weights were measured; after this, the dams were allowed to nurse them. The rate of still births (%) [(number of stillbirths/total number of pups delivered) x 100], the birth rates [(number of surviving pups/total number of pups delivered) x 100], external abnormality rates (%) [(number of pups with external abnormalities/number of surviving pups) x 100], and the sex ratios [number of males/(number of males+number of females)] were calculated. The stillbirths (except for the cases of marked post-mortem changes) were fixed in phosphate buffer 10 vol% formalin solution and stored. The surviving pups were observed once a day up to day 4 of nursing to see if they were still alive; the live-born survival rates (%) [(number of pups surviving on day 4 of nursing)/(number of live pups on day 0 of nursing) x 100] were calculated. Body weights of the live pups were measured on day 4 of nursing.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals in the main groups were sacrificed on day 42 of the study. All surviving animals of the satellite groups were sacrificed on day 14 of the recovery period.
- Maternal animals: All surviving animals in the main groups were sacrificed on lactation day 4. All surviving animals of the satellite groups were sacrificed on day 14 of the recovery period.

GROSS NECROPSY
- 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 numbers of corpora lutea and implantation sites of the female animals (dams) were counted. 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 / ORGAN WEIGHTS
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).

Postmortem examinations (offspring):

SACRIFICE
On lactation day 4, all pups were sacrificed by exsanguination under ether anesthesia and autopsied. The presence or absence of abnormalities in the organs and tissues, including the heads, chests, and abdomens were investigated.

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.

Reproductive indices:

After the end of the pre-mating administration period, the males and females of the same principal administration groups were allowed to cohabit overnight and the females which were found to form vaginal plugs or in which semen were observed in the vaginal smears the next morning were considered to have mated. The number of days required until mating were calculated, with the day the mating started as day 0. The mating rates (%) [(number of mated animals/number of cohabiting animals) x 100], the conception rates (%) [(number of fertilized females)/number of cohabiting females) x 100], and the fertilization rates (%) [(number of fertilized females)/(number of cohabiting males) x 100] were calculated from the mating results. The numbers of corpora lutea and the numbers of implantatios were counted and the implantation rates (%) [(number of implantations)/(number of corpora lutea) x 100] were calculated.

Offspring viability indices:

The rate of still births (%) [(number of stillborn/total number of pups delivered) x 100], the birth rates [(number of surviving pups/total number of pups delivered) x 100], the liveborn survival rates (%) [(number of pups surviving on day 4 of lactation)/(number of live pups on day 0 of lactation) x 100] were calculated.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality during the study period. No effects on the clinical signs were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No statistically significant differences in body weight were observed between the control groups and the treatment groups.

A significant decrease in food consumption was seen on day 42 in the males administered 250 mg/kg bw/d only. A significant increase was noted at a few time points in females administered 1000 mg/kg bw/d in the main group (gestation day 4) and the satellite group (administration day 25 and 29, recovery day 11). These variation are not considered to be treatment-related, as they were transient and no effect on body weight was observed.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test substance was administered by gavage daily, with doses based on the body weight, ensuring an accurate dosing of the animals.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There was no difference in the number and duration of estrous cycles between the control group and dose groups (see table 1).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- All 12 pairs of each dose group copulated successfully (copulation index 100%) (see table 3 and 4). One pair in the control group mated on day 7 after the mating period started and the other pairs mated within 4 days.
- All females became pregnant (fertility index: 100)
-The gestation index was 100% for all dose groups
- The delivery index was 100% for all dose groups
- All females delivered live pups

All the females delivered normally on days 21.1–22.5 of pregnancy, and no significant differences were observed between the control group and the treatment groups with respect to gestation periods, numbers of corpora lutea, implantation rates, stillbirth rates, number of live pups delivered, or live birth indexes (see table 2a and b).


ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant increases in the absolute kidney weight of the females in the 500 and 1000 mg/kg bw/d groups was observed. However, as the increase was not dose-related and only observed in one sex, 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/d was deemed to be incidental and thus not treatment-related. There were no effects on reproduction organs in either sex. The ovary weights were not recorded.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects were observed on reproductive organs and tissues, nor on other organs and tissues.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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 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 and there were no effects on reproductive organs and tissues.

OTHER EFFECTS (PARENTAL ANIMALS)

HAEMATOLOGY
A statistically significant decrease in the eosinophil level in the males administered 1000 mg/kg bw/d 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 (Petterino, 2006*), this is not considered to be an adverse effect of the treatment, but rather a reversible variation. In the 1000 mg/kg bw 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 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/d 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 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.

*Petterino, C. and Argentino-Storino, A. Clinical chemistry and haematology historical data in control
Sprague-Dawley rats from pre-clinical toxicity studies. Experimental and Toxicologic Pathology 2006; 57: 213–219

CLINICAL CHEMISTRY
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/d 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 effects on clinical chemistry parameters or histopathology were reported. No effects were observed in the satellite groups.

URINALYSIS (males)
There were no significant differences between the males in 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 test substance causes a higher urinary pH in exposed rats, but this effect is not considered to be adverse.

NEUROBEHAVIOUR
A statistically significant decrease in the number of rearings was observed in the males in the 1000 mg/kg bw/d 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/d 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.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
No significant differences were observed in viability of the pups between the control groups and the treatment groups. The viability on day 4 was similar in all groups (see table 2b).

BODY WEIGHT (OFFSPRING)
The body weight of the males in the 250 mg/kg bw/d group was significantly increased, compared to that of the control group, at the time of birth. As it was not dose-related, only observed in one sex and at one time point, it is not considered to be an adverse effect. No significant differences in body weights were observed between the control groups and the treatment groups on lactation day 4 (see table 5).

GROSS PATHOLOGY (OFFSPRING)
One live-born pup in the 1000 mg/kg bw/d group was born with a vestigial tail, however, due to the single incidence, this was not considered to be related to the treatment with the test substance. Apart from this, no unusual findings were noted during the gross necropsy of the pups that survived until lactation day 4.

OTHER FINDINGS (OFFSPRING)
A statistically significant difference in the sex ratio (No. males/No. liveborn pups) was observed in the 500 mg/kg bw/d group (0.44 versus 0.58 in the control group). As this effect was not seen in any other dose groups, it is not considered to be treatment-related (see table 5).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed up to and including the highest dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Estrous cycle in female rats during pre-mating period (main group)

		Estrous count
Dose (mg/kg bw)	No. animals	0	1	2	3	4	Mean ± SD	Mean duration of cycles Mean ±SD
0	12	0	0	0	5	7	3.6 ± 0.5	4.1 ± 0.3
250	12	0	0	0	5	7	3.6 ± 0.5	4.3 ± 0.4
500	12	0	0	0	1	11	3.9 ± 0.3	4.1 ± 0.1
1000	12	0	0	0	4	8	3.7 ± 0.5	4.1 ± 0.3

 

Table 2a: Delivery data on females in all groups

Dose (mg/kg bw)		No. pregnant females	Gestation period	No. of corpora lutea	No. of implantations	Implantation index
0	Total Mean SD	12	22.0 0.4	207 17.3 1.5	192 16.0 1.5	92.9 5.4
250	Total Mean SD	12	22.3 0.3	200 16.7 2.1	188 15.7 2.2	94.0 5.1
500	Total Mean SD	12	22.1 0.3	209 17.4 1.5	199 16.6 1.9	95.1 5.1
1000	Total Mean SD	12	22.2 0.4	201 16.8 2.2	193 16.1 1.8	96.4 5.9

 

Table 2b: Delivery data on females in all groups

Dose (mg/kg bw)		No. pregnant females	No. of stillborn (%)	No. of live born	Live birth index %	Viability index day 4 after birth
0	Total Mean SD	12	3 (1.5) (3.8)	183 15.3 1.3	98.5 3.8	97.9 3.1
250	Total Mean SD	12	1 (0.6) (1.9)	171 14.3 3.0	99.4 1.9	97.0 4.8
500	Total Mean SD	12	2 (1.0) (2.4)	178 14.8 2.9	99.0 2.4	98.1 2.9
1000	Total Mean SD	12	2 (1.1) (2.7)	182 15.2 1.7	98.9 2.7	98.0 5.3

Table 3: Mating and fertility data, males

 

Dose (mg/kg)	N	Days until copulation Mean ± SD	Copulation index (%)*	Insemination index (%)**
0	12	2.7±1.7	12/12 (100)	12/12 (100)
250	12	2.6±1.2	12/12 (100)	12/12 (100)
500	12	2.4±0.9	12/12 (100)	12/12 (100)
1000	12	2.8±1.1	12/12 (100)	12/12 (100)

* (No. of copulated animals/No. of mated animals) x 100

** (No. of males that inseminated females/No. of copulated females) x 100

 

 

Table 4: Mating and fertility data, females

 

Dose (mg/kg)	N	Days until copulation Mean ± SD	Copulation index (%)*	Fertility index (%)**
0	12	2.7±1.7	12/12 (100)	12/12 (100)
250	12	2.6±1.2	12/12 (100)	12/12 (100)
500	12	2.4±0.9	12/12 (100)	12/12 (100)
1000	12	2.8±1.1	12/12 (100)	12/12 (100)

* (No. of copulated animals/No. of mated animals) x 100

** (No. of pregnant animals/No. of copulated females) x 100

Table 5: Pup data

 

Dose (mg/kg bw)		Sex ratio	Body weight males	Body weight females
0	Total Mean SD	0.58	6.4 0.5	6.2 0.4
250	Total Mean SD	0.54	6.9* 0.5	6.5 0.5
500	Total Mean SD	0.44*	6.7 0.5	6.3 0.4
1000	Total Mean SD	0.55	6.7 0.4	6.3 0.4

* p<0.05

Applicant's summary and conclusion

Conclusions:

AEPD had no effect on the reproductive performance.

Executive summary:

A combined repeated dose and reproduction/developmental toxicity screening study performed according to OECD 422 to assess the potential reproductive and fertility effects of 2-amino-2-ethyl-1,3-propanediol (AEPD) is available (Ishida, 2004). In this study doses of 250, 500 and 1000 mg/kg bw/day of the test substance were administered by gavage to rats during pre-mating, mating, gestation and until lactation day 4. A satellite group with a recovery period of 14 days after dosing ended was included in the control and 1000 mg/kg bw/day groups. As no effects were observed up to and including the highest dose level, the NOAEL for systemic toxicity is considered to be>1000 mg/kg bw/day. In the P-generation, the parental reproduction parameters (including mating index, fertility index, gestation period, delivery index, and number of live pups) were not affected by treatment with the test substance up to and including the highest dose level of 1000 mg/kg bw/day. The NOAEL for reproduction is therefore considered to be >1000 mg/kg bw/day.