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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-amino-2-methyl-1,3-propanediol, AMPD, aminomethylpropanediol
- Analytical purity: 99.9% by weight
- Lot/batch No.: WF114801I1
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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD)
Sex:
female
Details on test animals 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

Administration / exposure

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)
Doses / concentrations
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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).

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproductive parameters

Group

Control

Treatment group 1

Treatment group 2

No. dams bred

6

5

7

No. (%) pregnant dams1

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 resorptions3

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

Applicant's summary and conclusion

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.