Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate

Endpoint summary

Description of key information

A 28 day oral toxicity study performed according to OECD Guideline 407 and under GLP with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6 is available, with reliability rating 1. Based on the absence of functional or morphological disturbances supporting the changes noted in the study, a NOAEL of > 1000 mg/kg was established.

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May - 15 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 159 grams; females: 133 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 18 May - 14 June 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for density of the test substance. The dose concentrations were corrected for a test substance purity of 40.4%.

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion treatment phase, according to a validated method (project 499169). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The purpose of this part of the study was to determine the accuracy of preparation, homogeneity and stability of the test substance in formulations.
For the formulation of Group 2, 3 and 4 prepared for use during treatment, the mean accuracy was outside the criterion of 90 – 110% of the target concentration (i.e. 86%, 76% and 89%). The mean accuracies were only slightly outside the criterion at the lowest (Group 2, i.e. 86%) and highest (Group 4, i.e. 89%) target concentration and were therefore considered acceptable. No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 d/w.

Remarks:

Doses / Concentrations:
0, 50, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 12-day dose range finding study with Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ßalaninate (project 499174: Based on the results of the range finding study, dose levels suggested for the main study (28 days toxicity study) were: 50, 300 and 1000 mg/kg body weight. No clinical signs were observed. Therefore, clinical observations in the main study were conducted immediately after dosing.).

Positive control:

Not required.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule:At least twice daily. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data

WATER CONSUMPTION:
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no evidence for dysfunction of organs

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male receiving 300 mg/kg died on scheduled day of necropsy between blood sampling and euthanasia. This was considered to be an accidental death and considered not to be related to treatment with the test substance. No clinical signs of toxicity were noted during the observation period. Salivation was incidentally noted in a single animal (on a single day). At the incidence observed, this finding was considered a sign of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

FOOD CONSUMPTION AND FOOD EFFICIENCY
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment. Statistically significant changes in platelet counts in females at 50 and 1000 mg/kg were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Statistically significant changes in creatinine level in males at 1000 mg/kg was considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain and no corroborative effects were found at microscopic examination.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS
Thymus weight (absolute and relative to body weight) was found to be statistically higher in females at 1000 mg/kg compared to the control group. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain. In addition these findings did not show a clear dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included diaphragmatic hernia, nodules in epididymides, fluid in the uterus, nodules in clitorial gland, foci in thymus and discolouration of the thymus.

HISTOPATHOLOGY
Minor, non-adverse, treatment-related microscopic findings were recorded in thymus of females and mesenteric lymph nodes of males:
Thymus: A slight increase in incidence of lymphocytolysis at a minimal degree was recorded in 4/5 females at 1000 mg/kg. This is a common background finding, which showed no clear dose-relation and was also recorded at minimal degree in 1/5 control females and 2/5 females at 50 mg/kg and was therefore not considered an adverse effect of the test item.
Mesenteric lymph nodes: Lymphoid hyperplasia was recorded at a minimal degree in some males of all Groups (Group 1: 1/5 males, Group 2: 1/5 males, Group 3: 2/5 males and Group 4: 3/5 males).
Lymphoid hyperplasia was also recorded in 1/5 females of Group 1 (control). The minimal lymphoid hyperplasia in the mesenteric lymph nodes and the remaining microscopic findings recorded were within the normal range of background pathology encountered in Wistar(Han) rats of this age and strain.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested (NOAEL >= 1000 mg/kg)

Critical effects observed:

not specified

Conclusions:

Based on the absence of functional or morphological disturbances supporting the changes noted for Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate, a NOAEL of 1000 mg/kg was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is performed according to OECD 407 guideline and under GLP and has reliability rating 1. It is sufficient to cover the information requirements in Annex VIII.

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

A 28-day oral toxicity study according to OECD 407 has been performed on Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6. The dose levels tested were 1000 mg/kg/day, 300 mg/kd/day and 50 mg/kg/day. This study was performed according to the current OECD 407 and EU method B7 protocols, with full GLP compliance and a well-defined test substance. No adverse effects were found in the study. The histopathology showed that the statistically higher thymus weight seen in females at 1000 mg/kg were minor, non-adverse, treatment-related lymphocytolysis. Since lymphocytolysis is a common background finding and based on the incidence, degree and lack of clear dose-relation seen in this study, this finding was not considered to be an adverse effect of the test item.

The parental toxicity effects reported in as 2-generation reproduction study on rats (parental dosing for 16 weeks) are consistent with the effects reported in the other repeat dose studies on these primary amine alaninates. The liver changes are indicative of metabolic processes and the results are also consistent with alklyamines suggesting similar toxicological mechanisms

The minimal lymphoid hyperplasia in the mesenteric lymph nodes of the males and the remaining microscopic findings recorded were within the normal range of background pathology encountered in Wistar(Han) rats of this age and strain. No adverse toxicity findings were observed up to the highest dose level tested, and a NOAEL value could therefore not be defined. The highest dose tested is therefore reported as NOAEL value for these this endpoint, since the use of ">" is not possible in the IUCLID filed specified for effect level.

 

The data from the 28 day oral toxicity study is therefore used to determine the DNEL values for oral, inhalation and dermal exposure following the ECHA guidance document for deriving DNEL values. As appropriate DNEL values can be calculated using the oral dosing study data, and due to the low vapour pressure of the substance, it is not justified on animal welfare grounds to perform repeat dose dermal or inhalation toxicity studies.

Work on analogue substances reviewed include further repeat dose oral testing (including dosing for reproduction tests) and these confirm low chronic toxicity with effects generally linked to liver function and some effects on kidneys. These have been considered in some cases to be adaptive and indicate metabolic processes.

This EPA study is considered valid in terms of grouping of substances. Examination of data on various primary alkylamines show little difference in repeat toxicity effects, including reproduction. Read-across is considered valid as the results are consistent with the findings in the more recent 28 days study (key study); this study show more adverse effects and is therefore considered a suitable surrogate for the reporting of reproductive effects.

The similarity in findings with the work on primary amines further justifies read-across to these potential metabolites. This is reviewed in the assessment report attached in Section 13.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The only available 28-day oral toxicity study on Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, is performed according to OECD 407 guideline under GLP conditions and has reliability rating 1. No adverse effects were seen in any of the selected doses in the study, and a NOAEL value could therefore not be defined. The highest dose tested is therefore reported as NOAEL value for this endpoint and used for DNEL derivation, since the use of ">" is not possible in the IUCLID filed specified for effect level below.  

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

There is no repeat dose inhalation study Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, however the substance is a paste with a low vapour pressure of < 1.5 mPa at 20°C. Significant exposure to vapours would not be expected at ambient temperatures so it is considered to not be scientifically valid to conduct a repeat dose inhalation study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

There is no repeat dose inhalation study Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, however the substance is a paste with a low vapour pressure of < 1.5 mPa at 20°C. Significant exposure to vapours would not be expected at ambient temperatures so it is considered to not be scientifically valid to conduct a repeat dose inhalation study.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

There is no repeat dose dermal study Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, but a 28 day oral toxicity study according to OECD 407 is available. Based on the physicochemical properties of the substance, it is considered very unlikely that dermal absorption would exceed oral absorption, so it would be expected than the oral NOAEL would be lower than a corresponding value from a dermal study. Results from the two acute toxicity tests available on the substance show no differences in toxicological profile, LD50 is > 2000 mg/kg bw for both exposure routes. Data from the repeat dose oral study can be used in the setting of DNELs in accordance with the REACH guidelines. As appropriate DNEL values can be calculated using the oral dosing study data, it is not justified on animal welfare grounds to perform a repeat dose dermal toxicity study.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

There is no repeat dose dermal study Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, but all three acute skin irritation / corrosion studies and the one acute dermal toxicity study in combination with the 28 day oral toxicity study indicates low toxicity of the substance. This does not justify the requirement for an additional dermal animal study to establish a local NOAEL for dermal exposure.

Justification for classification or non-classification

The EU CLP (GHS) criteria for classification for Specific Target Organ Toxicity (STOT) are based on data from a 90 day study, for Category 2 the range for such effects is 10-<100 mg/kg/day. Where the data are from a 28 day study these levels are multiplied by three. However there were no indications of any specific systemic toxic effects such as serious organ damage in any of the dose groups of Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6. Based on the lack of findings from the available 28 day oral toxicity study, there is no evidence of relevant specific target organ toxicity in the rats dosed at up to the highest dose level of 1000 mg/kg/day. Therefore it does not have to be classified and has no obligatory labelling requirement according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), nor the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.