Potassium N,N-dimethylglycinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-27 to 2015-07-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 Mar 1996)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test) (Jul 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium N,N-dimethylglycinate

EC Number:

241-629-2

EC Name:

Potassium N,N-dimethylglycinate

Cas Number:

17647-86-8

Molecular formula:

C4H8NO2.K

IUPAC Name:

potassium 2-(dimethylamino)acetate

Details on test material:

- Substance type: organic salt
- Physical state: white solid
- Expiration date of the batch: 04 Sept 2016
- Storage condition of test material: at room temperature (highly hygroscopic substance)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: individually in polycarbonate cages type III (floor area of about 800 cm²)
- Diet: ad libitum, ground Kliba maintenance diet mouse-rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, deionized water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test substance preparations were produced twice weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in deionized water over a period of 4 days at room temperature was proven before the start of the study. Samples of the test substance preparations were sent to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations. Three samples (one from the top, middle and bottom) were taken from the preparation vessels with the magnetic stirrer running.

Duration of treatment / exposure:

males: 29 days
females: 53 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1500, 4000, 12000 ppm
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1592, 4246, 12739 ppm
Basis:
other: content of the test substance (94.2 g/100 g)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters checked in table [No. 1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period and during administration period once a week

FOOD CONSUMPTION: Yes
- Time schedule: once weekly
- Exemptions:
Food consumption was not determined during the mating period (male and female F0 animals).
Food consumption of the F0 females with evidence of sperm was determined for GD 0 - 7, 7 - 14 and 14 - 20.
Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 4.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice a week
- Exemptions:
Water consumption was not determined during the mating period (male and female parental animals).
Additionally, during gestation (animals with evidence of sperm plugs) water consumption of the females were determined for GDs 0-1, 3-4, 6-7, 9-10, 12-13, 15-16 and 18-19 and during lactation period (animals with litter) for PNDs 0-1 and 3-4.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, at least 16 hours
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters checked in table [No. 2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once
- Animals fasted: Yes, at least 16 hours
- How many animals: first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters checked in table [No. 3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: once
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, at least 16 hours
- Parameters checked in table [No. 4] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration
- Dose groups that were examined: all (first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group)
- Parameters checked in table [No. 5, 6, 7] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see table 8
HISTOPATHOLOGY: Yes, see table 9

Statistics:

Means and standard deviations of each test group were calculated. In addition other analysis were performed for some parameters.
- Food consumption, water consumption, body weight and body weight change: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means.
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (twosided) for the hypothesis of equal medians
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
- Urinalysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same values of the dose group and the control, no statistical test is performed.
- Urine pH, volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians.
- Urine color and turbidity: Urine color and turbidity are not evaluated statistically.
- Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died or was sacrificed moribund during the study period. No test substance-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
Male animals of test group 3 (12000 ppm) showed a slight significant lower body weight on pre-mating days 7 (-6.0 %) and 13 (-5.0 %). The body weight change in male animals of test group 3 (12000 ppm) was decreased (-90.4 %) during pre-mating between day 0 and 7. The overall value for pre-mating body weight change was decreased significantly (-42.3 %). No further alterations of the body weight were observed after the first two weeks of administration in males. These findings were considered as treatment-related, but based on the degree of change as well as on its transient character they were assessed as not adverse.
Body weights and body weight changes were not significantly affected in female animals during pre-mating, mating, post-mating and gestation.

FOOD CONSUMPTION
Food consumption was significantly decreased (-23.7 %) in male animals of test group 3 (12000 ppm) during pre-mating between study day 0 and 7. No further effects were seen in food consumption. This finding was considered as treatment-related, but based on the degree of change as well as on its transient character it was assessed as not adverse.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
During pre-mating water consumption was significantly decreased between study day 0 and 3 in male animals of test group 3 (12000 ppm, -47.2 %) and in female animals of the same test group 3 between study day 0 and 3 (-29.9 %) as well as between study day 10 and 13 (-29.3 %). Whereas no effect can be seen in both sexes after the first two weeks of administration, it can be concluded, that the reason for this finding was assessed to be caused by the taste of the test item in the highest concentration, which the animals get accustomed to. So these findings were considered as treatment related, but not adverse. Mean daily substance intake can be found under "Any other information on results incl. tables."

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed. In males of test group 2 (4000 ppm) total protein values were lower compared to controls, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
No test substance related findings were seen during home cage and open field observations. One male animal of test group 3 (12000 ppm) and 1 male of control group (0 ppm) showed very frequent vocalizations when touched. This parameter is assessed as being spontaneous in nature and not related to treatment. Therefore no test substance-related effects were observed during sensorimotor test/reflexes. The overall motor activities (summation of all intervals) in male animals of test groups 3 (12000 ppm) and 2 (4000 ppm) was statistically significant decreased. Regarding the single motor activity intervals no statistically significant test substance-related deviations to the control were noted for male and female animals of test groups 1-3 (1500, 4000 and 12000 ppm). Since no treatment-related findings were observed in the functional observation battery, no comparable alteration observed in females and the overall comparable profiles of habituation between all dose groups and control, the observations of overall decreased motor activities in males were assessed as treatment-related but not adverse.

ORGAN WEIGHTS
Absolute organ weights: When compared to control group 0 (set to 100 %), the mean absolute weight of the heart was significantly decreased in females. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Relative organ weights: All mean relative weight parameters did not show significant differences when compared to the control group 0. The significant decreases of the absolute heart weights were not clearly dose-dependently. In test groups 1 (0.704 g) and 3 (0.694 g), they were below the historical control range (0.708 - 0.912 g), but without a histopathological correlate. Relative weights were not changed and were within the range of the historical control values. Therefore, the significant decreases of absolute weight of the heart was judged as incidental.


GROSS PATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

731 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponds to 12000 ppm (the highest tested dose)

2

Dose descriptor:

NOAEL

Effect level:

960 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponds to 12000 ppm (the highest tested dose)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results

Mean daily test-substance intake (mg/kg bw/d)

	Test group 1 (1500 ppm)	Test group 2 (4000 ppm)	Test group 3 (12000 ppm)
F0 males (premating)	112	276	731
F0 females (premating)	133	352	960
F0 females (gestation)	175	457	1265
F0 females (lactation)	206	548	1407

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weight of the heart was significantly decreased in females (statistically significant changes printed in bold):

	Female animals
Test group	Test group 1 (1500 ppm)	Test group 2 (4000 ppm)	Test group 3 (12000 ppm)
Heart	92 %*	96 %	90 %**

*p <= 0.05; **p <= 0.01