Potassium N,N-dimethylglycinate

Endpoint summary

Description of key information

The no observed adverse effect level (NOAEL) for general systemic toxicity was 731 mg/kg bw/d for male and 960 mg/kg bw/d for female Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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

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

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

Remarks:

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

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

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.

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.

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)

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)

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

731 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose toxicity of the test substance was examined in a test according to OECD guideline 422. The test substance was administered daily as solutions to groups of 10 male and 10 female Wistar rats (F0 animals) via the drinking water. The nominal concentrations of the used deionized water based formulation of the test item were 1592 ppm in test group 1, 4246 ppm in test group 2 and 12739 ppm in test group 3 corresponding to 1500, 4000 and 12000 ppm of test substance based on a content of 94.2 % of the test substance given in the used batch. Control animals were administered with the vehicle only (deionized water). The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to the day of scheduled sacrifice of the animals.

The parents' and the pups' state of health was checked each day and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly, males during 2 weeks of premating and females before and after the mating period, as well as in dams during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation (days 1 - 4). Water consumption of the F0 parents was determined twice weekly during premating. In dams water consumption was determined for gestation days 0 - 1, 3 - 4, 6 - 7, 9 - 10, 12 - 13, 15 - 16, 18 - 19 and lactation days 0 - 1 and 3 - 4. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day of parturition (PND 0) and on postnatal days (PND) 1 and 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (1500, 4000 and 12000 ppm) during the entire study. Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the test substance of 12000 ppm. Regarding 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.

Under the conditions of this study, the oral administration via drinking water of the test substance to Wistar rats revealed no signs of general systemic toxicity in male and female parental animals up to a concentration of 12000 ppm (731 mg/kg bw/d in males and 960 mg/kg bw/d in females). Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 12000 ppm in male and in female parental animals (731 mg/kg bw/d in males and 960 mg/kg bw/d in females).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP and guideline compliant study report.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.