Potassium N,N-dimethylglycinate

Endpoint summary

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:

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)

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 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): all animals were housed individually in polycarbonate cages type III (floor area of about 800 cm²)

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

Parental animals: Observations and examinations:

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.

OTHER: Haematology, clinical chemistry, urinalysis, neurobehavioural examination

Oestrous cyclicity (parental animals):

Not observed.

Sperm parameters (parental animals):

Parameters examined in all P male parental generations:
testis weight, epididymis weight, stages of spermatogenesis

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births (on PND 0), postnatal mortality (on PND 0; between PND 1 - 4) , clinical symptoms (including gross-morphological findings), body weight (on PND 1 and on PND 4)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals on study day 29
- Maternal animals: All animals on study day 53

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special intention was being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2, 3] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age under isoflurane anesthesia with CO2.
- These animals were subjected to postmortem examinations macroscopic and/or microscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. All organs were assessed macrospcopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.

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, gestation days: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means.
- Reproductive and offspring viability indices: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions.
Mating days until day 0 pc, % postimplantation loss, pups stillborn, % perinatal loss: Pair-wise comparison of the dose group with the control group using the WILCOXON test (onesided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.
- Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians.
- % live male day x, % live female day x: Comparison of the dose group with the control group was performed using the WILCOXON test (two-sided) for the hypothesis of equal medians.

Reproductive indices:

male/female mating index, male/female fertility index, gestation index, postimplantation loss

Offspring viability indices:

viability index, live birth index

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No animal died or was sacrificed moribund during the study period. No test substance-related clinical sgns were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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 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.

TEST SUBSTANCE INTAKE VIA DRINKING WATER (PARENTAL ANIMALS)
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".

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The stages of spermatogenesis in the testes of males in test group 3 (12000 ppm) were comparable to those of the controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Males: For parental males, which were placed with females to generate F1 pups, mating was confirmed. The male mating index was 100 % in all dose groups (1-3; 1500, 4000 and 12000 ppm). Male animals of control group showed a mating index of 90 %. Fertility was proven for most of the parental males within the scheduled mating interval to produce F1 litter (please refer to table 1 under "Any other information on results incl. tables"). One male of test group 3 (12000 ppm, No. 36 mated with female No. 136), two males of test group 2 (4000 ppm, Nos. 26 and 28 mated with female No. 126 and 128) and one male of the control group (0 ppm, No. 10 mated with female No. 110) did not generate implants in utero. One male of control group (0 ppm, No. 9 mated with female No. 109) did not generate F1 pups. The male fertility index was 90 % in test group 3 (12000 ppm) and control group, 80 % in test group 2 (4000 ppm) and 100 % in test group 1 (1500 ppm).
Females: The female mating index calculated after the mating period for F1 litter was 100 % in all dose groups (1-3; 1500, 4000 and 12000 ppm). Female animals of control group showed a mating index of 90 %. The mean duration until sperm was detected (GD 0) was 2.0, 2.2, 3.4, 2.5 days in test groups 0 - 3. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
All sperm positive rats got pregnant with one exception in test group 3 (12000 ppm) and two exceptions in test group 2 (please refer to table 2 under "Any other information on results incl. tables"). Female animal No. 136 (test group 3) which was mated with male No. 36, female No. 126 (test group 2) which was mated with male No. 26 and female No. 128 (test group 2) which was mated with male No. 28 did get sperm in vaginal smear but showed no implants. The mean duration of gestation was similar in all test groups (22.0 days in test group 0; 22.3 days in test groups 1, 22.0 days in test group 2, and 21.9 days in test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within or nearby the range of the historical control data.
The gestation index was 89 % in test group 0, and 100 % in test groups 1 - 3 (1500, 4000 and 12000 ppm). Also in the historical control data not always 100 % of the pregnant females had live pups. Consequently, the historical control data ranges from 70 % to 100 %. Therefore, the finding did reflect the normal range of biological variation inherent in the strain of rats used for this study. The postimplantation loss was 17.8 % in control group, 6.1% in test group 1 (1500 ppm), 12.4 % in test group 2 (4000 ppm) and 6.4 % in test group 3 (12000 ppm). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range (0.7 % - 18.3 %) of the historical control data.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.

OTHER FINDINGS (PARENTAL ANIMALS)
No treatment related changes were observed for hematological, urinanalysis or clinical chemistry parameters.

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) for systemic and reproductive toxicity

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) for systemic and reproductive toxicity

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam was evenly distributed about test groups 0 - 3. The respective values reflect the normal range of biological variation inherent in the strain used in this study. Each one stillborn pup was found in the litter of dams Nos. 133 and 138 of test group 3 (12000 ppm). This incidence were within the normal range of the historical control data.
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100.0 % (test groups 1 and 3) and 99.0% (test groups 0 and 2) based on 1 pup of the control group (0 ppm) was found dead and one pup of test group 2 (4000 ppm) was missing (cannibalized). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The rate live birth indices were 100 % in test groups 0, 1 and 2 (0, 1500 and 4000 ppm). In test group 3 (12000 ppm) the live birth index was 98.1 %. Two pups were stillborn in test group 3 (12000 ppm), each one in the litter of dams No. 133 and No. 138. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4. However, in test group 2 (4000 ppm) one female pups could be assessed only until PND 1 because it was missed (cannibalized) on PND 2 and in test group 3 (12000 ppm) one male and one female pup from different litters could not be assessed because they were stillborn on PND 0.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. One female runt was seen in test group 0 (0 ppm) and two female runts were seen in test group 2 (4000 ppm). These values were within the range of the biological variation inherent in the strain of rats used for this study.

GROSS PATHOLOGY (OFFSPRING)
One male pup of test group 3 (12000 ppm) and one female pup of control group (0 ppm) showed post mortem autolysis. Furthermore, in test group 2 (4000 ppm) one female pup could not be assessed because it was missing (cannibalized). These findings were assessed as being spontaneous in nature and thereby not related to treatment. No other findings were seen in any pup of any test group.

Reproductive effects observed:

not specified

Table 1: Male fertility indices

	Test group 0 (0 ppm)	Test group 1 (1500 ppm)	Test group 2 (4000 ppm)	Test group 3 (12000 ppm)
Male fertiltiy index [%]	90	100	80	90

* p ≤ 0.05; ** p ≤ 0.01

Table 2: Female fertility indices

	Test group 0 (0 ppm)	Test group 1 (1500 ppm)	Test group 2 (4000 ppm)	Test group 3 (12000 ppm)
Female fertility index [%]	100	100	80	90

* p ≤ 0.05; ** p ≤ 0.01

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Table 3: Pup sex ratio

PND 0	Test group 0 (0 ppm)	Test group 1 (1500 ppm)	Test group 2 (4000 ppm)	Test group 3 (12000 ppm)
Live males [%]	54.4	49.3	46.1	52.0
Live females [%]	45.6	50.7	53.9	48.0
PND 4
Live males [%]	54.7	49.3	46.5	52.0
Live females [%]	45.3	50.7	53.5	48.0

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

731 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive 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. 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. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (1500, 4000 and 12000 ppm) during the entire study. Regarding developmental toxicity, no signs of toxicity were observed in male or female parental animals of all test groups (1500, 4000 and 12000 ppm) during the entire study.

Under the conditions of this study, the oral administration via drinking water of Potassium N,N-dimethylglycinate to Wistar rats revealed no signs of reproductive or developmental toxicity. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 12000 ppm (731 mg/kg bw/d) in male and 12000 ppm (960 mg/kg bw/d) in female Wistar rats. The NOAEL for developmental toxicity in the F1 progeny was 12000 ppm.

Short description of key information:
The NOAEL for reproductive performance and fertility was set to 12000 ppm for male (731 mg/kg bw/d) and female (960 mg/kg bw/d) Wistar rats. The NOAEL for developmental toxicity was also set to 12000 ppm.

Justification for selection of Effect on fertility via oral route:
GLP and guideline compliant study report

Effects on developmental toxicity

Description of key information

The NOAEL for reproductive performance and fertility was set to 12000 ppm for male (731 mg/kg bw/d) and female (960 mg/kg bw/d) Wistar rats. The NOAEL for developmental toxicity was also set to 12000 ppm.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study report

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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: about 11-12 weeks (males and females)
- Housing: individually in polycarbonate cages type III
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): ad libitum, deionized water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- 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 mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

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

Maternal examinations:

WATER CONSUMPTION
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. During gestation and lactation no significant findings were observed.
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.
BODY WEIGHT DATA
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.

Fetal examinations:

Pup number and status at delivery
The mean number of delivered F1 pups per dam was evenly distributed about test groups 0 - 3. The respective values reflect the normal range of biological variation inherent in the strain used in this study. Each one stillborn pup was found in the litter of dams Nos. 133 and 138 of test group 3 (12000 ppm). This incidence were within the normal range of the historical control data.
Pup viability/ mortality
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100.0% (test groups 1 and 3) and 99.0% (test groups 0 and 2) based on 1 pup of the control group (0 ppm) was found dead and one pup of test group 2 (4000 ppm) was missing (cannibalized). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Pup clinical observations
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4. However, in test group 2 (4000 ppm) one female pups could be assessed only until PND 1 because it was missed (cannibalized) on PND 2 and in test group 3 (12000 ppm) one male and one female pup from different litters could not be assessed because they were stillborn on PND 0.
Pup body weight data
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. One female runt was seen in test group 0 (0 ppm) and two female runts were seen in test group 2 (4000 ppm). These values were within the range of the biological variation inherent in the strain of rats used for this study.
Pup necropsy observations
One male pup of test group 3 (12000 ppm) and one female pup of control group (0 ppm) showed post mortem autolysis. Furthermore, in test group 2 (4000 ppm) one female pup could not be assessed because it was missing (cannibalized). These findings were assessed as being spontaneous in nature and thereby not related to treatment. No other findings were seen in any pup of any test group.

Statistics:

Kruskal-Wallis Test; Wilcoxon-Test

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration via drinking water of Potassium N,N-dimethylglycinate 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).
The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 12000 ppm (731 mg/kg bw/d) in male and 12000 ppm (960 mg/kg bw/d) in female Wistar rats. The NOAEL for developmental toxicity in the F1 progeny was 12000 ppm.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Dose descriptor:

NOAEL

731 mg/kg bw/day

Study duration:

subacute

Species:

rat

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 reproductive 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. 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. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (1500, 4000 and 12000 ppm) during the entire study. Regarding developmental toxicity, no signs of toxicity were observed in male or female parental animals of all test groups (1500, 4000 and 12000 ppm) during the entire study.

Under the conditions of this study, the oral administration via drinking water of Potassium N,N-dimethylglycinate to Wistar rats revealed no signs of reproductive or developmental toxicity. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 12000 ppm (731 mg/kg bw/d) in male and 12000 ppm (960 mg/kg bw/d) in female Wistar rats. The NOAEL for developmental toxicity in the F1 progeny was 12000 ppm.

Justification for selection of Effect on developmental toxicity: via oral route:
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 reproduction/developmental toxicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.