Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 Aug - 04 Oct 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report that meets basic scientific principles.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not applicable
Principles of method if other than guideline:
The potential of DMAMP to cause toxicity to fertility and embryonic death, was assessed in a non-guideline screening study. Female rats were administered DMAMP from before mating until gestation day 14. During the pre-mating period the groups were administered increasing doses from 100 up to 1000 mg/kg bw/day (34 days). From Day 15 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 was counted and uteri of females lacking visible implantations were examined for signs of pregnancy (early resorption).
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: 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: 190.4 ± 8.2 g (mean ± SD)
- Housing: the animals were housed 2-3 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
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
At least 35 days (20 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)
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: gradually increased from 100 to 1000
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 DMAMP 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. 1000 mg/kg bw/day, which the rats were administered from study day 15, was considered to be the limit dose.
Positive control:
not applicable
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. After the 24-hour collection, samples were placed in the -80 °C freezer prior to analyses for the parent compound
Oestrous cyclicity (parental animals):
not evaluated
Sperm parameters (parental animals):
not evaluated
Litter observations:
not evaluated
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.
Postmortem examinations (offspring):
not evaluated
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. Also, the pre- and post-implantation losses were appropriately calculated. Other than this, statistical analyses was not performed for any of the parameters.
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.
Offspring viability indices:
not applicable
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
potential effect on implantation loss
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality. 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 the treatment group.

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 1). 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

With the exception of one control female, all females successfully bred with a pregnancy rate of 100% and there were no litter losses in the treatment group. There was no difference in the total number of implants in the treatment group when compared to controls. However, there was a 15.4% pre -implantation loss in DMAMP treated dams compared to 7.1% in controls, and a 16.8% post-implantation loss in DMAMP treated dams compared to 5.4% in controls.

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 DMAMP were proportional to the administered dose.
Dose descriptor:
LOEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Potential effect on implantation
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Until further data become available
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Not applicable
Reproductive effects observed:
not specified

Table 1: Reproductive parameters

Group

Control

DMAMP

No. dams bred

6/7

7/7

No. (%) pregnant dams1

6/6 (100.0%)

7/7 (100.0%)

Mortality

0/7

0/7

No. moribund dams

0/7

0/7

No. aborted

0/6

0/7

No. delivered early

0/6

0/7

No. litters totally resorbed

0/6

0/7

No. litters with normal embryos

6/6

7/7

Corpora lutea/dam (mean ± SD)

15.8 ± 2.1

15.9 ± 1.9

Implantations/dam (mean ± SD)

14.7 ± 1.8

13.1 ± 5.6

Mean pre-implantation loss (%)2

7.1 ± 6.0

15.4 ± 35.0

Resorptions/litter (mean ± SD)3

0.8 ± 0.8

2.4 ± 3.2

Resorptions/litters with resorptions3

1.3 (5/4)

3.4 (17/5)

Mean post-implantation loss (%)4

5.4 ± 4.5

16.8 ± 24.5

Normal embryos/litter (mean ± SD)

13.8 ± 1.3

10.7 ± 5.7

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: Individual animal data

 

Control – Animal no.

Corpora lutea count

Implantation count

Pre-implantation loss

Resorption/s count

% Pre-implantation loss

% Post-implantation loss

Normal Embryos 

7391

15

15

0

1

0.0

6.7

 14

7392

16

14

2

1

12.5

7.1

 13

7393

20

18

2

2

10.0

11.1

 16

7394

15

13

2

1

13.3

7.7

 12

7395

14

14

0

0

0.0

0.0

 14

7396

Not pregnant

 

7397

15

14

1

0

6.7

0.0

 14

Average

15.8

14.7

1.2

0.8

7.1

5.4

 13.8

SD

2.14

1.75

0.98

0.75

0.06

0.04

 1.3

Treated – Animal no.

Corpora lutea count

Implantation count

Pre-implantation loss

Resorption/s count

% Pre-implantation loss

% Post-implantation loss

 Normal Embryos

7405

13

13

0

0

0.0

0.0

 13

7406

15

15

0

1

0.0

6.7

 14

7407

18

1

17

0

94.4

0.0

 1

7408

17

17

0

4

0.0

23.5

 13

7409

17

17

0

2

0.0

11.8

 15

7410

17

16

1

1

5.9

6.3

 15

7411

14

13

1

9

7.1

69.2

 4

Average

15.9

13.1

2.7

2.4

15.4

16.8

 10.7

SD

1.86

5.61

6.32

3.21

35.5

24.5

 5.7

Table 3: Historical control data*

Study No./year

Mean percent pre-implantation loss

Mean percent post-implantation loss

1 / 2005

11.0 ± 17.0

8.2 ± 12.6

2 / 2005

6.5 ± 9.9

4.4 ± 4.9

3 / 2008

10.8 ± 12.5

1.0 ± 2.5

4 / 2009

6.8 ± 12.2

5.2 ± 6.9

5 / 2005

4.9 ± 11.4

1.2 ± 3.7

6 / 2005

4.2 ± 6.4

7.0 ± 8.3

7 / 2005

6.6 ± 8.6

7.1 ± 9.9

8 / 2006

12.3 ± 15.4

7.8 ± 10.6

9 / 2009

5.6 ± 12.7

4.1 ± 7.8

10 / 2010

3.6 ± 5.4

1.9 ± 4.3

11 / 2010

3.6 ± 5.8

1.4 ± 2.8

* Data from developmental toxicity probe studies (study 1-4) and developmental toxicity studies (study 5-11)

 conducted in CD rats

Conclusions:
There were no treatment-related effects on clinical observations, body weight, body weight gains, or feed consumption of females administered DMAMP at dose levels up to 1000 mg/kg/day during the prebreeding and gestation phases of the study. There was a 15.4% preimplantation loss in DMAMP treated dams compared to 7.1% in controls, and a 16.8% postimplantation loss in DMAMP treated dams compared to 5.4% in controls. These values for DMAMP treated dams were slightly outside of historical control ranges for both preimplantation (3.6 ± 5.8 – 12.3 ± 15.4) and postimplantation (1.0 ± 2.5 – 8.2 ± 12.6) loss. Due to the limited design of this non-guideline screening study with only one dose and low animal numbers, it is not possible to conclude whether this was a chance finding or was treatment-related.
Executive summary:

Groups of seven non-pregnant female Crl:CD(SD) rats were administered a vehicle control and the test material (DMAMP) daily, by gavage, at dose levels of 0 (control) or 100 mg/kg/day. The dose level of each 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), through breeding (up to 7 days) and until GD 14. 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 following the first dose at 250 mg/kg/day (DMAMP).

There were no treatment-related effects on clinical observations, body weight, body weight gains, or feed consumption of females administered DMAMP at dose levels up to 1000 mg/kg/day during the pre-breeding and gestation phases of the study. There was a slight increase in both pre-implantation and post-implantation loss in dams administered 1000 mg/kg/day of DMAMP above control and historical controls. Due to the limited design of this non-guideline screening study with only one dose and low animal numbers, it is not possible to conclude whether this was a chance finding or was treatment-related.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A subchronic toxicity study was performed in rats according to OECD 408, using DMAMP adjusted to pH 9 (Wasil, 2012). 10 rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day by gavage for 90 days. The weight of the reproductive organs and tissues (ovaries, uterus, epididymides, testes) was comparable between the control and all treatment groups. No treatment-related effects were seen during the gross pathology and microscopic histopathology examinations of the reproductive organs and tissues (coagulating glands, cervix, epididymides, ovaries with oviducts prostate, seminal vesicles, testes, uterus, vagina) at the highest dose level of 1000 mg/kg bw/day.

The potential of 2-(dimethylamino)-2-methyl-1-propanol (DMAMP) to cause toxicity to fertility and embryonic death in rats was assessed in a non-guideline screening study (Rasoulpour and Andrus, 2011). 7 female Crl:CD(SD) rats/dose were administered DMAMP (adjusted to pH 9) from before mating until gestation day 14. During the pre-mating period the groups were administered an initial 100 mg/kg bw/day dose that increased every 3-7 days up to the highest dose of 1000 mg/kg bw/day (34 days in total). From Day 15, all the rats were administered 1000 mg/kg bw/day. The rats were sacrificed on gestation day 14 and the number and position of implantations, viable embryos, and resorptions were recorded. In addition the number of corpora lutea was counted and uteri of females lacking visible implantations were examined for signs of pregnancy (early resorption). No treatment-related clinical signs were observed during the study period. The body weight and food consumption was similar in the treatment group compared with the control group. No treatment-related findings were noted in the reproductive tract during the gross pathology.
6/6 dams in the control group and 7/7 dams in the treatment group were pregnant after mating.
It should be noted that the correct number of corpora lutea cannot be ascertained based on the design of the study as the oestrus cycle starts again on day 4 post-partum when the number of corpora lutea is usually determined. Therefore, the correct evaluation of the pre-implantation loss is not ensured. There was a 15.4% preimplantation loss in DMAMP treated dams compared to 7.1% in controls, and a 16.8% postimplantation loss in DMAMP treated dams compared to 5.4% in controls. These values for DMAMP treated dams were slightly outside of historical control ranges for both preimplantation (3.6 ± 5.8 – 12.3 ± 15.4) and postimplantation (1.0 ± 2.5 – 8.2 ± 12.6) loss. Due to the limited design of this non-guideline screening study with only one dose and low animal numbers, it is not possible to conclude whether this was a chance finding or was treatment-related.  

2014 OECD guideline 421 study:

The purpose of this study was to evaluate the potential effects of 2-Dimethylamino-2-methyl-1-propanol (DMAMP) on reproductive function and prenatal/early neonatal growth and survival of the offspring. Groups of 10 male and 10 female Crl: CD(SD) rats were administered DMAMP daily by oral gavage in Milli Q- purified water at dose levels of 0 (control), 50, 250, or 1000 mg/kg/day. The control and dose solutions were adjusted to approximately pH 9 and were administered at a dose volume of 4 ml/kg body weight. Females were dosed once daily for two weeks prior to breeding and through breeding (up to two weeks), gestation (three weeks), lactation (four days), and until necropsy. Pups were euthanized on postpartum day 4. Females were necropsied on postpartum days 5-7. Males were dosed for two weeks prior to breeding and continuing through breeding until necropsy (test day 31). Effects on reproductive function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues emphasizing organs of the reproductive system. In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed.

There were no treatment-related effects in clinical observations, body weights and body weight gains, feed consumption, reproductive function, prenatal/early neonatal growth and survival of the offspring, or gross pathology in either sex at all dose levels tested. 

Treatment-related effects of DMAMP were limited to the 1000 mg/kg/day dose group and included: decreased relative and absolute epididymal weights in males and histopathological observations in the kidneys of both sexes. The slightly decreased epididymal weights were not accompanied by a histopathological correlate and were considered non-adverse. 

Histopathological observations in the kidney of rats of the 1000 mg/kg/day group included very slight to slight hyperplasia (focally extensive or multifocal) of the pelvic transitional epithelium, with accompanying very slight to slight chronic active inflammation. In addition, one female had moderate focally extensive necrosis of the transitional epithelium of the renal papilla, and accompanying moderate chronic active inflammation.

Based on histopathological alterations of the kidney, the no-observed-effect level (NOEL) for general toxicity was 250 mg/kg/day. The NOEL for reproductive effects was 1000 mg/kg/day, the highest dose level tested.

Short description of key information:

An OECD guideline 421 study is available.

Justification for selection of Effect on fertility via oral route:

Acceptable, well documented study report that meets basic scientific principles.

Effects on developmental toxicity

Description of key information

An OECD guideline 421 study is available.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2015 - 3 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted under GLP.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
Test animals:
- Source: Charles River (Raleigh, North Carolina)
- Age and weight at study initiation: Sexually mature adult weighing approximately 200-250 g
- Fasting period before study: none
- Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form
- Water: municipal water was provided ad libitum (water was analyzed periodically for chemical parameters and biological contaminants)
- Acclimation period: 4 days
- Housing: after assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
- Temperature: 22°C with a range of 20°C-26°C
- Humidity: 50% with a range of 30-70%
- Air Changes: 10-15 times/hour (average)
- Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Ultrapure water
Details on exposure:
All dosing solutions were prepared by mixing the test material in Ultrapure water adjusted to approximately a final pH of 9, at concentrations of 0, 25, 75, or 250 mg/ml and administered at a dose volume of 4 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. The control rats were dosed with Ultrapure water adjusted to approximately a final pH 9 at 4 ml/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration verification of all dose solutions and homogeneity of the low- and high-dose solutions were determined pre-exposure. The method used for analyzing the test material in Ultrapure water was high performance liquid chromatography with positive ion electrospray ionization and tandem mass spectrometry detection operating in the multiple reaction monitoring mode (HPLC/ESI-MS/MS) (LCMS.
Details on mating procedure:
Sexually mature, adult virgin females were naturally mated with males of the same strain (one male:one female) at the suppliers facility. Females were checked for in situ copulation plugs the following morning, and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier and maintained in the study record. Rats arrived in the laboratory on GD 1 or 2.
Duration of treatment / exposure:
Days 6 - 20 of gestation
Frequency of treatment:
Once daily
Duration of test:
Gestation days: 6 - 21
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
24
Control animals:
yes
Details on study design:
Dose levels for this study were selected on the basis of the reproduction/developmental toxicityscreening study. Gavage exposure to DMAMP at doses up to 1000 mg/kg/day resulted in no treatment-related effects in clinical observations, body weights and body weight gains, feed consumption, reproductive function, prenatal/early neonatal growth and survival of the offspring, or gross pathology in either sex. The high-dose of 1000 mg/kg/day represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any toxicity that was observed among the high-dose group rats and to establish a no-observed-effect level (NOEL).
Maternal examinations:
A cage-side examination was conducted twice daily, approximately at the same time each day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. All animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Ovaries and uterine content:
On GD 21, all surviving females (not fasted) were sedated with a mixture of isoflurane vapors and medical oxygen, euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.

The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative portions of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of the liver, kidneys, and gross lesions was not conducted. Transponders were removed and placed in with the preserved tissues.

A detailed examination of the reproductive tract was performed, and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicated a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations was stained with a 10% aqueous solution of sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

The sex and body weight of all viable fetuses were recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods based on Trueman et al. (1999) and Zablotny (2002). A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination. However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination would have provided more meaningful data about a suspected abnormality.

All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment group assignment.
Fetal examinations:
NOTE: all references to tables can be found in the original report.

The incidence of external, craniofacial, visceral and skeletal variations and malformations observed among fetal rats is summarized in Table 10, while individual litter data are reported in Appendix Table 8. Compared to controls, there were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups. The small number of alterations observed in fetuses from dams administered DMAMP either occurred at low frequencies and/or were not dose related. Details of these findings are described below.

External Examination
There were no treatment-related external alternations in any dose group. Incidental findings bearing no relationship to treatment included the e malformations aglossia, microagnathia, and gastroschisis umbilicus (Text Table 7). Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Craniofacial Examination
There were no craniofacial alternations in any dose group.

Visceral Examination
There were no treatment-related visceral alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformations situs inversus, misshapen heart, and persistent 4th aortic arch (Text Table 8) and the variations fused lung, missing caudal lung lobe, hemorrhage liver, pale liver, supernumerary hepatic liver lobule, hemorrhage adrenal, bifurcated renal vein, and accessory blood vessel kidney. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Skeletal Examination
There were no treatment-related skeletal alternations in any dose group. In the 100 and 300 mg/kg/day dose groups there was a statistically -identified decrease in the incidence of delayed ossification (DO) of the thoracic centra compared to control (Text Table 9). This finding bore no toxicological significance as the incidence was lower than controls. Other incidental findings bearing no relationship to treatment included the malformation missing sternebrae (Text Table 10) and the variations supernumerary bone skull, DO parietal, DO occipital, DO thoracic centra, DO sternebrae, irregular pattern of ossification sternebrae, calloused ribs, class I and II wavy ribs, DO lumbar centra, and extra 1st lumbar rib. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Statistics:
Maternal body weights, maternal body weight gains, maternal organ weights (absolute and relative), gravid uterine weights, fetal body weights, and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with
Bonferroni's correction (Miller, 1966) was performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations and litter size were evaluated using a non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios
were analyzed using a binomial distribution test. Females lacking visible implantations or totally resorbed litters at the scheduled necropsy were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha = 0.02; Grubbs, 1969) and, if excluded, were excluded for sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
See effects levels above.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
See effects levels above.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The No-Observed-Effect Level (NOEL) is 300 mg/kg bw /day for both maternal and embryo/fetal toxicity. Based on these findings, it is not appropriate to classify this substance as a reproductive toxicant.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of 2-(dimethylamino)-2-methyl-1-propanol (DMAMP) in Crl:CD(SD) rats following repeated gavage administration. Groups of 24 time-mated female rats were administered DMAMP in Ultrapure water by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses.

Maternal toxicity was limited to dams given 1000 mg/kg/day and consisted of treatmentrelated decreases in body weight, body weight gain and feed consumption during the treatment period compared to controls. There were no effects on body weight, body weight gain, or feed consumption in the 100 and 300 mg/kg/day dose groups. At necropsy there were no treatment-related gross pathological observations, and there were no treatment-related differences in kidney or liver weights for any of the treated groups when compared to their respective controls.

There were no treatment-related fetal malformations in any group up to, and including, 1000 mg/kg/day, the highest dose tested. In the 1000 mg/kg/day dose group there was a treatment-related decrease in fetal body weights (male 6.8%, female 5.8%, and combined 6.6%) compared to controls. There were no effects on fetal body weights in the 100 and 300 mg/kg/day dose groups.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 300 mg/kg/day, and the embryo/fetal NOEL was 300 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1) and is thus sufficient to fulfill the standard information requirements set out in Annex VII, 8.5 of Regulation (EC) 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The potential embryotoxicity of 2-(dimethylamino)-2-methylpropan-1-ol (DMAMP) was assessed in a Micromass test (limb bud micromass assay, similar to methodology validated by ECVAM and published as ‘Micromass Test, method of Brown, INVITTOX No. 122’) (Ellis-Hutchings and Marshall, 2011). Undifferentiated rat embryo limb bud mesenchymal cells were isolated from rat embryos on gestation day 13 and exposed to the test substance in vitro. After 5 days, the cells had differentiated into chondrocytes and the effect of the test substance on specific parameters (cell differentiation, cell viability, cell number, neutral red uptake, cell growth) was measured. The ID50 (50% inhibition of cell differentiation and number of foci) and IC50 (50% inhibition of cell viability and growth) were determined and indicated that DMAMP is a non-embryotoxic substance.

In order to meet the standard information requirements according to Regulation (EC) 1907/2006 Annex IX, Column I, 8.7.2, a GLP-compliant prenatal development toxicity study according to OECD 414 is required. As no additional data are available, a prenatal developmental toxicity study following OECD 414 in rats via the oral route is proposed.

The available data on the reproduction, fertility and developmental toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

Justification for selection of Effect on developmental toxicity: via oral route:

This is a current guideline study

Justification for classification or non-classification

The available data on the reproduction, fertility and teratogenic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.