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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:
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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 2-(dimethylamino)-2-methyl-1-propanol, DMAMP
- Analytical purity: 99.7% (by area)
- Lot/batch No.: 201000687-30

Test animals

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

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
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)
Doses / concentrations
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

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. 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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

Not applicable

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

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 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.