2-amino-2-methylpropane-1,3-diol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug - 04 Oct 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles

Principles of method if other than guideline:

The potential of AMPD to cause developmental toxicity, specifically embryonic death, was assessed in a non-guideline screening study. Female rats were administered AMPD from before mating until gestation day 14. During the pre-mating period the groups were administered either increasing doses from 100 up to 1000 mg/kg bw/day (34 days) or 1000 mg/kg bw/day, the limit dose (6 days). The rest of the exposure period all rats were administered 1000 mg/kg bw/day. The mortality, clinical signs, body weight, food consumption and urine content of test substance were reported. The rats were sacrificed on gestation day 14 and number and position of implantations, viable embryos, and resorptions were recorded. In addition the number of corpora lutea were counted and uteri of females lacking visible implantations were examined for signs of pregnancy (early resorption).

GLP compliance:

no

Remarks:

This non-guidleline screening study has been well conducted and reported. All raw data will be stored for 75 years in the archives of The Dow Chemical Company.

Limit test:

no

Specific details on test material used for the study:

- Test material: XU-12398.00
- Chemical name: 2-amino-2-methyl-1,3-propanediol
- Synonyms: AMPD, aminomethylpropanediol
- Supplier: ANGUS Chemical Company, Buffalo Grove, Illinois
- Lot #: WF114801I1
- Purity: non-GLP certificate of analysis lists the purity of the test material as 99.9% by weight.
- Molecular formula: C4H11NO2
- Molecular weight: 105.1
- CAS number: 115-69-5

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, Michigan, USA
- Age at study initiation: approximately 8 wks (females)
- Weight at study initiation: 188.8 ± 5.9 g (mean ± SD, group 1) and 239.6 ± 15.5 (mean ± SD; group 2, from study day 29)
- Housing: the animals were housed two-three per cage in stainless steel cages during the acclimation period. After study start, animals were housed singly in stainless steel cages, except during breeding (one male to one or two females). Cages had wire mesh floors and were suspended above catch pans. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet and also provided environmental enrichment. In order to better visualize copulation and plugs, gauze was not placed in the cages during the mating period.
- Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri, USA) in meal form, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 with a tolerance of ± 1 °C and maximum permissible excursion of ± 3 °C
- Humidity (%): 40-70
- Air changes (per hr): 12-15 (average)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose suspensions/solutions were prepared at least biweekly during the study, and were not corrected for purity of the test material. As all test materials are basic in nature, the dose suspensions/solutions were adjusted to pH 9 for animal welfare concerns, and were mixed overnight and maintained on a stir plate.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1-2
- Length of cohabitation: up to 7 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly, in wire mesh cages

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Group 1: at least 32 days (17 days pre-mating, up to 7 days during mating, GD 0-14)
Group 2: at least 21 days (6 days pre-mating, up to 7 days during mating, GD 0-14)

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 12 weeks (females) and sexually mature (males)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The initial dose level of AMPD was 100 mg/kg bw/day and increased every 3-7 days to 250, 500, 750 and 1000 mg/kg bw/day, respectively, during the pre-mating period, providing the dose was well-tolerated. A dose of 1000 mg/kg bw/day, which the rats were administered from study day 18, was considered to be the limit dose. Due to mortality (2/7 rats) in group 1, a second group (7 females) was administered 1000 mg/kg bw/day from 6 days prior to breeding until gestation day 14.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily during the study period
- Cage side observations included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, fecal/urinary quantity, mortality, morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, and assessment of general behavior, injuries or palpable mass/swellings.
- Time schedule: daily during the exposure period, approximately 1 hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: at least once during the pre-exposure period and daily prior to dosing throughout the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OTHER:
Urine samples were collected from all the females in group 1 only, starting immediately after the first dose of 100 mg/kg bw/day and, again on Day 4, following the first dose of 250 mg/kg bw/day. The rats were held in matabolism cages and the urine was collected on dry ice overnight (approximately 24 hours). The animals had access to water and food.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving animals were sacrificed on gestation day 14

GROSS NECROPSY
- No gross necropsy to record treatment-related effects was perfomed. A detailed examination of the reproductive tract; the number and position of implantations, viable embryos, and resorptions were recorded, and the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status. The liver was preserved in neutral, phosphate-buffered 10% formalin, and transponders were removed and placed in jars with the livers.

HISTOPATHOLOGY / ORGAN WEIGHTS
No histopathological examinations were performed.

Statistics:

Means and standard deviations were calculated for all continuous data. Feed consumption values were excluded from analysis if the feed was spilled or scratched. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969). The percent of pre- and post-implantation loss were calculated.

Reproductive indices:

Pregnancy rate = (number of females with visible implantations / number of females mated) x 100

Pre-implantation loss* = (No. corpora lutea-implantations/ No. corpora lutea) x 100

Post-implantation loss* = (No. implantations – viable embryos / No. implantations) x 100

* Percent pre- and post-implantation loss was determined for each litter, followed by calculation of the mean of these litter values. Data for pre- and post-implantation loss did not undergo statistical analyses, but were interpreted within the context of the current and historical control values.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality (2/7 rats) in group 1, due to a gavage error. Therefore, a second group (7 females) was administered 1000 mg/kg bw/day from 6 days prior to breeding until gestation day 14.

Body weight and weight changes:

no effects observed

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:

not examined

Haematological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

With the exception of one control female, all females successfully bred with a pregnancy rate of 100%. Every litter from all treatment groups contained embryos that were considered normal for gestational age (GD 14) with no total litter losses. There was no difference in the total number of implants in any treatment group when compared to controls. There were no treatment-related effects on preimplantation or postimplantation loss in dams administered AMPD (groups 1 & 2) at 1000 mg/kg/day.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
2/7 females in group 1 died due to a gavage dosing error during the pre-mating period. No treatment-related clinical signs were observed during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No differences in body weight and food consumption were observed between the control group and treatment group 1 and 2, respectively.

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 PERFORMANCE (PARENTAL ANIMALS)
- All the pairs in the treatment groups copulated successfully (copulation index 100%) (see Table 2). In 1/7 dams in the control group, no signs of successful copulation was observed.
- All the females that copulated successfully became pregnant (fertility index: 100)
- No litters were aborted, delivered early or totally resorbed
- All the litters had normal embryos

No differences were observed between the control group and the treatment groups with respect to numbers of corpora lutea, implantation rates, resorption rates, pre-implantation loss, post-implantation loss and number of normal embryos per litter (see Table 1).

GROSS PATHOLOGY (PARENTAL ANIMALS)
The examination of the reproductive tract did not reveal treatment-related effects on the number and position of implantations, viable embryos, resorptions or ovarian corpora lutea.

OTHER FINDINGS (PARENTAL ANIMALS)
The urinalysis indicated that the urine concentrations of AMPD were proportional to the administered dose (see Table 2).

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

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

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

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

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table 1: Reproductive parameters

Group	Control	Treatment group 1	Treatment group 2
No. dams bred	6	5	7
No. (%) pregnant dams^1	6/6 (100.0%)	5/5 (100.0%)	7/7 (100.0%)
Mortality	0/7	2/7	0/7
No. moribund dams	0	0	0
No. aborted	0	0	0
No. delivered early	0	0	0
No. litters totally resorbed	0/0	0/0	0/0
No. litters with normal embryos	6/6	5/5	7/7
Corpora lutea/dam (mean± SD)	15.8 ± 2.1	15.2± 1.3	15.6± 2.4
Implantations/dam (mean± SD)	14.7± 1.8	15.0± 1.6	15.4± 2.1
Mean pre-implantation loss (%)^2	7.1± 6.0	1.4± 3.2	0.7± 1.9
Resorptions/litter (mean± SD)^3	0.8± 0.8	0.6± 0.9	0.7± 1.0
Resorptions/litters with resorptions^3	1.3 (5/4)	1.5 (3/2)	1.7 (5/3)
Mean post-implantation loss (%)^4	5.4± 4.5	4.0± 5.8	4.7± 6.2
Normal embryos/litter (mean± SD)	13.8± 1.3	14.4± 1.8	14.7± 2.3

¹No. females with visible implantations/total No. bred

²Mean %/litter (calculated as [(No. corpora lutea - implantations) / No. corpora lutea] x 100)

³Not statistically analysed

⁴Mean %/litter (calculated as [(No. implantations – normal embryos) / No. implantations] x 100)

Table 2: Concentrations of test substance in urine

Group	Animal	Measured concentration (µg analyte/g urine)
		Sample set 1*	Sample set 2**
Control	10A7391	< LLQ***	< LLQ
	10A7392	< LLQ	< LLQ
	10A7393	< LLQ	< LLQ
	10A7394	< LLQ	< LLQ
	10A7395	< LLQ	< LLQ
	10A7396	< LLQ	< LLQ
	10A7397	< LLQ	< LLQ
1	10A7412	948	3599
	10A7413	617	3462
	10A7414	1362	3219
	10A7415	900	3201
	10A7416	655	2087
	10A7417	1115	2596
	10A7418	580	2504
	Mean	833	2953
	SD	289	561

* Sample set 1 = samples collected on treatment day 1 for group 1 (administered 100 mg/kg bw/day)

** Sample set 2 = samples collected on treatment day 4 for group 1 (administered 250 mg/kg bw/day)

*** LLQ = Lowest Level Quantitated = 62.8 µg AMPD/g urine

Conclusions:

There were no treatment-related effects on clinical observations, body weight, body weight gains, or feed consumption of females administered AMPD at dose levels up to 1000 mg/kg/day during the prebreeding and gestation phases of the study. There were no treatment-related effects on evaluated reproductive parameters in dams administered 1000 mg/kg/day of AMPD.

Executive summary:

Groups of seven non-pregnant female Crl:CD(SD) rats were administered a vehicle control or AMPD daily, by gavage, at dose levels of 0 (control) or 100 mg/kg/day. The dose level of the test group was increased periodically to escalate up to 250, 500, 750 or 1000 mg/kg/day until either the maximum tolerated dose (MTD) or the limit dose (1000 mg/kg/day) was reached. The females were maintained at 1000 mg/kg/day prior to breeding (13 to 20 days depending on the test material), through breeding (up to 7 days) and until GD 14. An additional group of seven female rats was administered 1000 mg/kg/day AMPD (AMPD-2 group) for six days prior to breeding, through breeding (up to 7 days) and until GD 14. This group was added following the loss of two females from the original AMPD group (AMPD-1 group) due to gavage error during the prebreeding phase. Body weights, feed consumption and clinical observations were evaluated daily prior to breeding and periodically during gestation. On GD 14, the females were euthanized, and the reproductive tract was examined for the number of corpora lutea, conceptuses, and implantations. Additionally, urine was collected for toxicokinetic analyses following the first dose at 100 mg/kg/day and again on day 4 or following the first dose at 250 mg/kg/day (AMPD 1). There were no treatment-related effects on clinical observations, body weight, body weight gains, or feed consumption of females administered AMPD at dose levels up to 1000 mg/kg/day during the prebreeding and gestation phases of the study. There were no treatment-related effects on evaluated reproductive parameters in dams administered 1000 mg/kg/day of AMPD.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Not indicated in the abstract.

GLP compliance:

not specified

Limit test:

no

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)

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crj:CD(SD)IGS

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: 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

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:

After the end of the dosage period before mating, males and females of the same dose group of the main group were housed together overnight. Evidence of copulation was obtained by the presence of sperm or a vaginal plug.

Mating index, female and male fertility index for each group were calculated according to the following formula.
- Mating index (%) = (Number of mated animals/number of animals cohabitated) X 100
- Female fertility index (%) = (Number of pregnant females/number of sperm-positive females) X 100
- Male fertility index (%) = (Number of pregnant females/number of males cohabitated) X 100

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:

Duratiion: for males, 42 days. For females, from 14 days before mating to day 4 of lactation.

Frequency of treatment:

Daily

Details on study schedule:

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.

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:

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. The dosage route was selected was oral administration.1000 mg/kg bw/day is the recommended maximum dose according to OECD test guideline 422. The administration period was for males from 14 days prior to mating and through the mating period up to the day before autopsy (42 days dosage) , and for females 14 days prior to mating and mating period and pregnant period through lactation day 4 (42 to 48 days dosage). The recovery period was 14 days following completion of dosage administration

Positive control:

Not applicable.

Parental animals: Observations and examinations:

General clinical observations. Open field examination.

Oestrous cyclicity (parental animals):

Vaginal smear was collected and microscopically examined for all female animals in the main group, every day from the start day of dosage to until copulation was recognized. The vaginal smear image during the dosage period before mating was classified into proestrus, estrus, metestrus, and anestrous.

Sperm parameters (parental animals):

The presence or absence of sperms in the vaginal smear was studied during the mating period.

Litter observations:

The stillborn (excluding the case of significant changes after death) was fixed and retained in phosphate-buffered 10 vol% formalin. The observation of life and death of the offspring was made once every day until lactation day 4 and the probability of survival was calculated by the following formula:
Probability of survival of offspring (%) = (Number of live pups on lactation day 4 / number of live pups on lactation day 0) X 100.

After the body weight was measured on lactation day 4, all cases under ether anesthesia were fatally exsanguinated, the autopsy was performed, and presence or absence of abnormalities of organs and tissues including the head, the chest, and the abdomen were studied. Moreover, the individual body weight of offspring was measured and the average value was calculated by male and female by each unit.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:]
- The number of live pups and the number of the stillborn were counted on the day of birth.
- Presence of absence of abnormalities in the offspring
- Determination of sex
- Body weight measurement

In addition, the rate of the stillborn, the birth rate, the rate of external surface abnormality, and the sex ratio were calculated.
- Rate of the stillborn (%) = (Number of the stillborn/ total number of birth) X 100
- Birth rate (%) = (Number of live pups/ total number of offspring) X 100
- Rate of external surface abnormality (%) = (Number of offspring with external surface abnormality / total number of live pups) X 100
- Sex ratio = Number of males/number of males + number of females

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

Statistics:

Reproductive parameters were statiistically evalueated by the Bartlett test (significance level 0.01, both sides), Dunnett test in the case of equal variance, and Dunnett mode test (significance level of 0.05 and 0.01, both sides) in the case of unequal variance. As for birth weight (by male and female), test the equal variances by the Bartlett test, Dunnett test in the case of equal variance, and Dunnett mode test (significance level of 0.05 and 0.01, both sides) in the case of unequal variance, after determining the average value of each dam animal.

Reproductive indices:

Gestation period (day) = Lactation day 0 – gestation day 0
Gestation index (%) = (Number of females delivering live pups/ number of pregnant females) X 100
Rate of implantation (%) = (Number of implantations/ number of corpora lutea) X 100

Offspring viability indices:

Rate of the stillborn (%) = (Number of stillborn / total number of pups) X 100
Birth rate (%) = (Number of live pups / total number of pups) X 100
Skeletal abnormalities (%) = (Number of pups with skeletal abnormalities / total number of live pups) X 100
Sex ratio (%) = (Number of males / (number of males + number of females) X 100
Survival of offspring (%) = (Number of live pups on lactation day 4 / number of live pups on day 0) X 100

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 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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, 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

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No test substance-related effects were observed on the estrous cycle, number of days until copulation, copulation index, insemination index, or fertilisation index. Furthermore, there were no effects of test article administration on the gestation period, birth index, numbers of corpora lutea, numbers of implantations, implantation index, parturition or nursing behavior, numbers of stillborn pups, still birth index, numbers of liveborn pups, liveborn index, sex ratio, observation of liveborn pups at birth or necropsy at 4 days postpartum, as well as body weights or viability.

Histopathological examination in the repeated dose study revealed cell infiltration in the mucosa of the forestomach and glandular stomach, erosion in the glandular stomach, increased numbers of globular leukocytes in the glandular stomach, thickening of the glandular stomach mucosa and thickening of the limiting ridge in males of the 1000 mg/kg group.

Open field examination revealed low values for rearing in males of the 1000 mg/kg group in weeks 3, 4, 5 and 6 of the administration.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

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

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:

not specified

Clinical signs:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the test substance did not show any effects on the reproductive parameters.

Key result

Reproductive effects observed:

no

Conclusions:

With regard to reproductive/developmental toxicity items, the test substance showed no adverse effects on any relevant parameters.

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.

No effect of the administration of the test substance on the estrous cycle, the number of days required for copulation, copulation rate, insemination rate, and conception rate. In addition, no effect of the administration of the test substance on the gestation period , birth rate, the number of corpora lutea, the number of implantation sites, the rate of implantation, delivery, lactation activities of dam animals, the rate of the stillborn, birth rate, sex ratio, and offspring, the autopsy findings postnatal day 4, weight, and viability. From these results, it was determined that the NOAEL for reproductive-developmental toxicity was 1000 mg/kg/day for both parental animals of both sexes and the off-spring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication/study report which meets basic scientific principles. The method is validated by ECVAM but is not available as a guideline.

Qualifier:

no guideline available

Principles of method if other than guideline:

The potential embryotoxicity of a test substance is assessed in the Micromass test (limb bud micromass assay) by exposing undifferentiated rat embryo limb bud mesenchymal cells to the test substance and measuring the effect on specific parameters (cell differentiation, cell viability, cell number, neutral red uptake, cell growth) when the cells have differentiated into chondrocytes. The limb bud cells are isolated from rat embryos on gestation day 13 and a single cell suspention is divided into wells on a 96-well plate. Cell medium with or without the test substance is added and the plates are incubated for 5 days. The ID50 (50% inhibition of cell differentiation and number of foci) and IC50 (50% inhibition of cell viability and growth) are determined and used to classify the test substance as non-embryotoxic, weak embryotoxic or strong embryotoxic substance. The method used in the Micromass test (limb bud micromass assay) was based on established techniques (Flint, O. P., and Orton, T. C. (1984). An In Vitro Assay for Teratogens with Cultures of Rat Embryo Midbrain and Limb Bud Cells. Toxicol. Appl. Pharm. 76, 383-95) and similar to methodology validated by ECVAM (Micromass Test, method of Brown, INVITTOX No. 122) and available on the website (http://ecvam.jrc.it).

GLP compliance:

no

Remarks:

This non-guidleline ex vivo/in vitro study has been well conducted and reported. All raw data will be stored for 75 years in the archives of The Dow Chemical Company.

Limit test:

no

Specific details on test material used for the study:

- Test material: XU-12398.00
- Chemical name: 2-amino-2-methyl-1,3-propanediol
- Synonyms: AMPD, aminomethylpropanediol
- Supplier: ANGUS Chemical Company, Buffalo Grove, Illinois
- Lot #: WF114801I1
- Purity: non-GLP certificate of analysis lists the purity of the test material as 99.9% by weight.
- Molecular formula: C4H11NO2
- Molecular weight: 105.1
- CAS number: 115-69-5

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (CRL) Inc., Portage, Michigan, USA
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 200-250 g (females)
- Housing: the animals were housed one per cage in stainless steel cages with wire mesh floors, suspended above absorbent paper. Non-woven gauze were placed in the cages to provide a cushion from the flooring for rodent feet. The cages contained a feed crock and a pressure activated lixit valve-type watering system. Enrichment for the rats included the use of a non-woven gauze pad per animal in caging, a stainless steel object in the cage for manipulation, and periodic rotation of each rack of cages in the animal room to allow animals a variety of visual experiences.
- Diet: LabDiet Certified Rodent Diet #5002 in pelleted form (PMI Nutrition International, St. Louis, Missouri, USA), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 1 weeks prior to scheduled Cesarean section

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 (maximum permissible excursion was 22 ± 3 °C)
- Humidity (%): 40-70
- Air changes (per hr): 12-15, on average
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: not applicable

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

other: not applicable

Analytical verification of doses or concentrations:

no

Details on mating procedure:

The cells used for the in vitro assay were isolated from the embryos of time-mated rats.

Duration of treatment / exposure:

The embryo cells used in the in vitro assay were exposed constantly for 96 h. The rats, from which the embryos were derived, were not exposed to the test substance.

Control animals:

other: not applicable

Details on study design:

Range-finding study:
The first experiment was performed with concentrations up to 1000 µM, as this was approximately ten times higher than the upper estimate of the achieved steady state serum concentration following in vivo limit dosing (1000 mg/kg bw/day). As 50% cytotoxicity was achieved the same dose range was selected for the second experiment.

Procedure for initiating the cell culture:
The cells were isolated by euthanising the rats by carbon dioxide inhalation on GD 13 and isolating the embyos aseptically from the uterus in sterile Hank’s Balanced Salt Solution (HBSS). Hind limb buds were then removed by microdissection, pooled and dissociated using 1.0% trypsin + 0.1% Ethylenediaminetetraacetic acid (EDTA) in calcium/magnesium free phosphate buffered saline (CMF). The resulting cell suspension was rinsed with culture medium (10% v/v FCS in Ham’s F12), passed through a 100 µm pore size nylon mesh cell strainer to remove clumps of cells, and assessed for cell viability and number of cells using 0.1% trypan blue and a hemocytometer. The cells were then re-suspended in culture medium to achieve a final concentration of approximately 2 x 10E7 viable cells/mL.

Procedure for exposure of the cells:
Aliquots of final cell suspensions were seeded onto 24-well surface-modified polystyrene cell culture plates (PrimeraTM, BD, New Jersey, USA) at 10 µL per spot (200,000 viable cells) and incubated at 37 ºC in an atmosphere of 5% CO2/95% humidified air for two hours to allow for cell attachment. Following attachment, each well was gently flooded with 1 mL culture medium with or without test material. AMPD was added directly to the culture media (10% v/v fetal calf serum (FCS) in Ham’s F12) following solubilization in a separate vehicle when necessary (≤ 0.05% v/v DMSO for the positive control atRA). Test and positive control solutions were prepared in the appropriate vehicle with the highest concentration pH adjusted to 7.2-7.4 prior to serial dilution. The plates were then returned to the incubator for the duration of the 5-day (approximately 96 h) static culture.

Number of replications:
3 replicates were used per concentration level in two independent experiments.

Controls:
All-trans-retinoic acid (atRA) was used as the positive control at concentrations of 0.000033, 0.00033, 0.0033, 0.01, 0.033, 0.33, 1 and 33 µM, previously found to induce approximately 10-100% inhibition in chondrocyte differentiation. Negative controls with culture medium and DMSO were included in both assays.

Cytotoxicity (total number of viable cells):
The cell viability was determined after approximately 96 h of culture. The treated culture media was carefully removed and the cells rinsed two times with saline. Following fixation of the cells with 2.5% glutaraldehyde for 20 minutes, cell viability was assessed via cellular uptake of neutral red. The cells were cultured in the presence of 0.005% neutral red in fresh culture media for 2-3 hours followed by removal of the media, three rinses with saline and acid alcohol elution of the neutral red from the viable cells. The absorbance of the eluted neutral red were then read at 540 nm using a multi-well plate spectrophotometer.

Measurement of differentiated chondrocytes:
Following removal of the acid alcohol eluate, each well was gently rinsed with saline and incubated overnight at room temperature with 1% alcian blue in 0.1N hydrochloric acid, a dye which stains cells rich in anionic glycoconjugates such as chondrocytes. The alcian blue solution was then removed and each well was gently rinsed with saline. Assessment of chondrocyte differentiation was determined by (1) manual quantitation of the stained chondrocyte foci and (2) eluate absorbance measurement at 620 nm using a multi-well plate spectrophotometer following elution of the alcian blue stain using freshly prepared 6M guanidine hydrochloride.

Data analysis to determine cell viability and growth:
Dose response curves were plotted as a percent of the vehicle control from the measured results (neutral red absorbance or number of alcian blue positive foci or alcian blue absorbance). Using non-linear regression analysis (curve fitting), the IC50diff and IC50cyt were calculated. The ratio of the IC50cyt to IC50diff values was compared. Interpretation of a positive response was evaluated in the context of the data using the two-fold rule and a linear discrimination analysis prediction model as follows:
A) 2-fold rule: Embryotoxicity potential would be suggested if the IC50diff value was less than half the IC50cyt value (Uphill et al., 1990)
B) Prediction model: Linear discrimination algorithm analysis was used to predict one of three embryotoxicity potentials: non-, weak, or strongly embryotoxic.

Function I 6.65 * log (IC50diff) – 9.49
Function II 6.16 * log (IC50diff) – 8.29
Function III -1.31 * log (IC50diff) – 1.42

If the result of function I exceeded the results of function II and III, then the chemical would be predicted to be non-embryotoxic; if the result of function II exceeded the results of function I and III, the chemical would be predicted to be weakly embryotoxic; if the result of function III exceeded the results of functions I and II, the chemical would be predicted to be strongly embryotoxic (Genschow et al., 2000 and 2002).

Maternal examinations:

General clinical observations. Open field examination.

Details on maternal toxic effects:

Maternal toxic effects:not examined

Key result

Dose descriptor:

other: not applicable

Basis for effect level:

other:

Remarks on result:

other: AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.

Key result

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Key result

Dose descriptor:

other:

Remarks on result:

other: AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Cytotoxicity/ Cell viability:

Table 1: Results of cytotoxicity and chodrocyte differentiation, average of 2 trials

Concentration (µM)	Cytotoxicity (% of control)		Chondrocyte differentiation, Foci Quantification method (% of control)		Chondrocyte differentiation, Alcian Blue Elution method (% of control)
AMPD	Mean ± SD	N	Mean ± SD	N	Mean ± SD	N
0.01	93.7 ± 13.6	6	92.5 ± 8.6	6	87.8 ± 15.7	6
0.1	84.1 ± 7.3	6	92.9 ± 8.6	6	92.5 ± 42.3	6
1.0	73.9 ± 20.9	6	84.4 ± 18.0	6	81.4 ± 22.4	6
10	77.0 ± 21.7	6	71.8 ± 19.4	5	81.8 ± 19.2	6
100	76.8 ± 17.4	6	69.4 ± 14.9	6	88.0 ± 25.9	6
1000	90.2 ± 42.5	6	81.3 ± 15.4	6	95.3 ± 10.2	6
	IC50cyt= 1000µM / NQ		IC50diff= NQ
atRA
0.000033	100.5 ± 20.3	6	92.3 ± 14.0	6	67.5 ± 17.5	6
0.00033	94.0 ± 13.1	6	92.8 ± 17.3	6	68.8 ± 31.9	6
0.0033	92.9 ± 9.0	6	79.0 ± 14.4	6	42.3 ± 14.2	6
0.01	101.4 ± 25.5	6	65.6 ± 25.6	6	31.5 ± 11.5	6
0.033	73.5 ± 34.3	6	33.1 ± 14.5	6	13.6 ± 15.6	6
0.33	66.9 ± 62.9	5	1.5 ± 3.6	6	14.6 ± 6.5	6
1.0	119.9 ± 6.7	3	0.0 ± 0.0	3	0.0 ± 9.2	3
33	18.7 ± 43.3	3	0.0 ± 0.0	3	0.0 ± 11.6	3
	IC50cyt= 500µM		IC50diff= 0.024µM

NQ = non-quantifiable due to lack of inhibition of cell viability or differentiation

In experiment 1, 50% cytotoxicity was observed at the highest concentration, 1000µM (individual well results not reported), while less than 30% cytotoxicity was evident in all concentrations in experiment 2 (Table 2, average of 2 experiments). Therefore, it was not possible to calculate the average IC50_cyt(50% inhibition of cell viability and growth).

It was not possible to apply the Foci Quantification method to determine the chondrocyte differentiation (IC50_diff), as the number of foci was > 50% at all tested concentrations. Determining the chondrocyte differentiation using the Alcian Blue Elution method was shown to be inaccurate when comparing the data to the results of manual count of foci in the culture. Therefore these results were disregarded. For all the cytotoxicity measurements the positive control reduced cell viability/differentiation by > 80%, and was therefore valid.

Embryotoxic potential:

 

Table 2: Analysis of embryotoxic potential, 2-fold rule method

	IC50diff(nM)	IC50cyt(nM)	IC50 ratio	Prediction
Run average
AMPD	1000000/NQ	1000000/NQ	1.0/na	Non-embryotoxic
atRA	24.25	500014	0.25	Embryotoxic
Run No. 1
AMPD	1000000	1000000	1.00	Non-embryotoxic
atRA	13.6	27.6	0.49	Embryotoxic
Run No. 2
AMPD	NQ	NQ	NQ	Non-embryotoxic
atRA	34.90	1000000	0.00003	Embryotoxic

NQ = non-quantifiable, an IC50 could not be calculated

 

In the 2 -fold rule method AMPD was predicted to be non-embryotoxic (see Table 2). The positive control was correctly predicted to be embryotoxic and therefore considered to be valid.

 

Table 3: analysis of embryotoxic potential, prediction model method

	IC50diff(µg/mL)	Function I	Function II	Function III	Prediction
Run average
AMPD	105.1/NQ	3.95/NQ	4.16/NQ	-4.07/NQ	Non-embryotoxic
atRA	0.0073	-24.01	-21.74	1.44	Strongly embryotoxic
Run No. 1
AMPD	105.1	3.9537	4.1631	-4.0683	Non-embryotoxic
atRA	0.0041	-25.37	-23.0	1.71	Strongly embryotoxic
Run No. 2
AMPD	NQ	NQ	NQ	NQ	Non-embryotoxic
atRA	0.0105	-22.65	-20.48	1.17	Strongly embryotoxic

NQ = non-quantifiable, an IC50 could not be calculated

In the prediction model method AMPD was predicted to be non-embryotoxic (see Table 3). The positive control was correctly predicted to be strongly embryotoxic and therefore considered to be valid.

Conclusions:

AMPD was predicted to be non-embryotoxic in the rat embryo limb bud micromass assay.

Executive summary:

The purpose of this study was to evaluate the embryotoxicity potential of 2-amino-2-methyl-1,3-propanediol (AMPD) in rats using the limb bud micromass assay. This preliminary screen involves high density culture of undifferentiated mesenchymal cells isolated from gestation day (GD) 13 rat embryo limb buds whereby during the 96 hour culture the cells proliferate, condense and differentiate into chondrocytes. Limb bud micromass cultures were exposed to six different concentrations of each test material. At the end of the culture period, the cells were assessed to determine the concentration at which 50% of the vehicle control group cell viability (IC50cyt) or chondrocyte differentiation (IC50diff) was inhibited. A neutral red updake assay was used for the cell viability/cytotoxicity evaluation. Inhibition of chondrocyte differentiation was assessed by staining the chondrocytes with alcian blue followed by 1) manually counting the stained foci, and 2) measurement of the alcian blue absorbance following elution from the foci. The embrotoxicity potential of AMPD was analyzed using two established methods (2-fold rule, prediction model) which assess the relative sensitivity for effects on differentiation versus cytotoxicity. Two independent trials were conducted for the test materials and a positive control inhibitor of chondrocyte differentiation, all-trans retinoic acid (atRA). Cell viability was marginally reduced following AMPD treatment (IC50cyt = 1000 μM). Inhibition of chondrocyte differentiation was marginally reduced following AMPD treatment. Due to limited or no inhibition of chondrocyte differentiation an IC50diff value could not be quantified for AMPD. Using the 2-fold rule and prediction model methods, AMPD was predicted to be non-embryotoxic. The known human and rat teratogen, atRA, was correctly predicted to be embryotoxic or strongly embryotoxic using the 2-fold rule or prediction model method, respectively.

Reference 2

Endpoint:

developmental toxicity

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.

Qualifier:

according to guideline

Guideline:

other: OECD 422 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

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)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Sprague-Dawley, Crj:CD(SD)IGS
- 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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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.

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.

Details on mating procedure:

After the end of the dosage period before mating, males and females of the same dose group of the main group were housed together overnight. Evidence of copulation was obtained by the presence of sperm or a vaginal plug.

Mating index, female and male fertility index for each group were calculated according to the following formula.
- Mating index (%) = (Number of mated animals/number of animals cohabitated) X 100
- Female fertility index (%) = (Number of pregnant females/number of sperm-positive females) X 100
- Male fertility index (%) = (Number of pregnant females/number of males cohabitated) X 100

Duration of treatment / exposure:

Duratiion: for males, 42 days. For females, from 14 days before mating to day 4 of lactation.

Frequency of treatment:

Dailly

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:

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. The dosage route was selected was oral administration.1000 mg/kg bw/day is the recommended maximum dose according to OECD test guideline 422. The administration period was for males from 14 days prior to mating and through the mating period up to the day before autopsy (42 days dosage) , and for females 14 days prior to mating and mating period and pregnant period through lactation day 4 (42 to 48 days dosage). The recovery period was 14 days following completion of dosage administration

Maternal examinations:

General clinical observations. Open field examination. Vaginal smear was collected and microscopically examined for all female animals in the main group, every day from the start day of dosage to until copulation was recognized. The vaginal smear image during the dosage period before mating was classified into proestrus, estrus, metestrus, and anestrous.

Clinical signs:

no effects observed

Description (incidence and severity):

No effects on clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study period.

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 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:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following findings were assumed to be incidental changes in terms of frequency of occurrence and pathological properties.

1) Analysis at the end of treatment:
Stomach: Dark reddish nest in grandular stomach was found in two cases of females in the 500 mg/kg treated group.
Testis: Miniaturization was found in one male case in the 250 mg/kg treated group.

2) Analysis at the end of recovery
No abnormalities were found among any animals.

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

Details on results:

No test substance-related effects were observed on the estrous cycle, number of days until copulation, copulation index, insemination index, or fertilisation index. Furthermore, there were no effects of test article administration on the gestation period, birth index, numbers of corpora lutea, numbers of implantations, implantation index, parturition or nursing behavior, numbers of stillborn pups, still birth index, numbers of liveborn pups, liveborn index, sex ratio, observation of liveborn pups at birth or necropsy at 4 days postpartum, as well as body weights or viability.

Histopathological examination in the repeated dose study revealed cell infiltration in the mucosa of the forestomach and glandular stomach, erosion in the glandular stomach, increased numbers of globular leukocytes in the glandular stomach, thickening of the glandular stomach mucosa and thickening of the limiting ridge in males of the 1000 mg/kg group.

Open field examination revealed low values for rearing in males of the 1000 mg/kg group in weeks 3, 4, 5 and 6 of the administration.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Conclusions:

With regard to reproductive/developmental toxicity items, the test substance showed no adverse effects on any relevant parameters.

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.

No effect of the administration of the test substance on the estrous cycle, the number of days required for copulation, copulation rate, insemination rate, and conception rate. In addition, no effect of the administration of the test substance on the gestation period , birth rate, the number of corpora lutea, the number of implantation sites, the rate of implantation, delivery, lactation activities of dam animals, the rate of the stillborn, birth rate, sex ratio, and offspring, the autopsy findings postnatal day 4, weight, and viability. From these results, it was determined that the NOAEL for reproductive-developmental toxicity was 1000 mg/kg/day for both parental animals of both sexes and the off-spring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available