trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

Endpoint summary

Description of key information

There is a new OECD442 repeat dose study with reproduction and developmental screening available. It is to the current OECD guideline and full GP compliant.

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:

09 October 2017 - 21 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: See "Principles of method if other than guideline"

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Purity/Composition correction factor: ~30% (29.7% w/w in water solution);
batch number: 48724
pH (20% aq. solution): 6.3 (6.0 - 7.0);
Specific gravity / density: 1.047 kg/m3 at 20°C.

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks (males) and 14 weeks (females)
- Weight at study initiation: 272 - 301 g (males) and 210 - 252 g (females)
- Fasting period before study: No (during motor activity measurements the animals had no access to food; males were fasted before necropsy overnight with a maximum of 24 hours, females were not fasted)
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal water, ad libitum
- Acclimation period: at least 8 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed. There were no known contaminants in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 32-57
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 Nov 2017 To: 31 Jan 2018

Route of administration:

oral: gavage

Details on route of administration:

Dose volume: 5 mL/kg bw

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. From start until Day 24 of treatment, the dosing formulations were prepared daily as a solution and dosed within 5 hours after completion of the preparation of the formulation. From Day 25 of treatment, the dosing formulations were prepared weekly in bulk, filled out in daily portions and stored at room temperature for a maximum of 8 days from preparation. Prior to dosing, the formulations were stirred for at least 30 minutes. All formulations were used within the demonstrated stability period of 8 days of preparation.
Adjustment was made for specific gravity of the test item. A factor of 3.4 was used to correct for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure. Dose formulation samples were collected in the first week of treatment for concentration analysis (all groups), and for homogeneity and stability analysis (low and high dose groups formulations). The homogeneity results obtained from the top, middle and bottom for the low and high dose group preparations were averaged and utilized as the concentration results. Duplicate sets of samples (approximately 500 mg) for each sampling time point were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Stability of the test item in water was determined after 5 hours and 8 days storage at room temperature.

Duration of treatment / exposure:

Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 60-70 days). Females that failed to deliver pups were treated for 40-42 days.

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Initially, the dose range finder was started by treatment of two groups for 10 consecutive days, i.e. Group 1 at 500 mg/kg bw/day and Group 2 at 1000 mg/kg bw/day. As the results were considered to be inconclusive for selecting dose levels for the main study, an additional group was treated at 750 mg/kg bw/day for 25 consecutive days. Each group consisted of 3 females.
The following parameters were included:
Mortality: Twice daily throughout the study.
Clinical Observations: At least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body Weights: On Day 1 prior to dosing and on Days 5 and 10. For Group 3 the body weights were determined every 5 days from Day 1 onwards.
Food Consumption Over Days 1-5 and 5-10. For Group 3 food consumption was determined over each of these 5-day periods between body weight measurements.
All animals were subjected to an external, thoracic and abdominal examination on Day 10 for groups 1 and 2 and on day 26 for group 3 after the last observation of clinical signs (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only (overnight with a maximum of 24 hours), females were not fasted)
- How many animals: All
- All parameters according to guidelines were examined (including thyroid hormone (males only) and coagulation parameters (prothrombin time and activated partial thromboplastin time)).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only (overnight with a maximum of 24 hours), females were not fasted)
- How many animals: All
- All parameters according to guidelines were examined.

URINALYSIS: No

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Week 4 of treatment (males) and during the last week of lactation (i.e. PND 9, 10, 11 and 12; females)
- Dose groups that were examined: 5/sex
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (mean of three measurements per animal), locomotor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER:
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
- Reproduction and developmental toxicity parameters are included in sections 7.8.1 and 7.8.2.

Sacrifice and pathology:

GROSS PATHOLOGY: All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Main organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (all animals)
For males that failed to sire (except for males which were selected) and females that failed to delivery pups in addition cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina were examined histopathologically.
For the remaining animals gross lesions and masses were examined.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the arena observations in this study. Chromodacryorrhoea was noted in one male at 150 mg/kg bw/day on one day of the premating period. Rales were observed in one control male, one female at 150 mg/kg bw/day and one female at 500 mg/kg bw/day. This clinical sign was considered to be of no toxicologically relevance as it was temporary observed in single animals per group and because of its equal distribution over the dose groups (including controls). Salivation seen after dosing in two males at 150 mg/kg bw/day and among all male and female animals at 500 mg/kg bw/day was considered to be a physiological response (possibly due to the taste of test item) rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Any other clinical signs noted during the treatment period (i.e. scabs and alopecia) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
On day 2 of treatment, one female dosed at 50 mg/kg bw/day started showing breathing difficulties during dosing and died a few moments later. Macroscopic examination revealed liquid in the lungs. Based on these finding its death was considered the result of a dosing error and not related to the test item. After its death, this female was replaced by one of the reserve females. Initiation of treatment of the replacement female was on Day 2.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight gain of treated male and female rats remained in the same range as concurrent controls over the treatment period. It should be noted that body weight gain in one control male and one female at 500 mg/kg bw/day was deviating from normal over (part of) the treatment period. For the male, a normal weight gain was observed for the first two weeks of the study, followed by a minimal body weight loss (of approximately 3%) over the remaining two week study period. For the female, a low level of body weight gain was observed during the last part of its gestation period. Nevertheless it delivered a normal litter, and its growth during lactation was normal. Based on the single incidences of deviating growth, these findings were considered of no toxicological relevance and not related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted over the treatment period.
It should be noted that a low food consumption was observed for one female at 500 mg/kg bw/day during the last part of its gestation period, which was concurrent to its low body weight gain.

Food efficiency:

no effects observed

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

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, increased counts for neutrophils, and a concurrent decreased count for lymphocytes were observed in plasma of male and female animals, achieving levels of statistical significance for neutrophils in both sexes (absolute counts in males and as percentage of WBC in females) and for percentage of lymphocytes in females when compared to controls. As a consequence, increased neutrophils-to-lymphocytes ratios were observed for males and females at 500 mg/kg bw/day when compared to controls and the animals treated at lower doses (neutrophils-to-lymphocytes ratio's of 0.14, 0.13, 0.15 and 0.32 (males), 0.42, 0.41, 0.38 and 0.79 (females) for control rats and rats dosed at 50, 150 and 500 mg/kg bw/day, respectively). Since an increased neutrophils-to-lymphocytes ratio is considered a marker for (systemic) inflammation, a relation of the changes in these ratios to the findings in the mesenteric lymph nodes could not be ruled out, but could not be established in the current study.

Coagulation parameters of treated rats were considered not to be affected by treatment. The statistical significance in prothrombin time (PT) noted for males at 50 mg/kg bw/day when compared to controls had occurred by chance. In the absence of a dose-related trend this change was considered not related to treatment and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment. The statistical significances apparent for creatinine levels in males at 150 and 500 mg/kg bw/day were considered the result of relatively lower creatinine values in control males, rather than indicative of a treatment-related effect. Also in the absence of a clear dose response relationship, no toxicological significance was attached to this finding. Any other statistically significant changes in clinical biochemistry parameters, i.e. sodium in males at 50 and 150 mg/kg bw/day and alanine aminotransferase in females at 50 mg/kg bw/day, were considered not to be related to treatment as these occurred in the absence of a dose-related trend.
Serum levels of T4 in males were considered not to be affected by treatment. A relatively low group mean value for T4 was observed in males at 500 mg/kg bw/day in comparison with that in controls. Since all individual T4 values of the 500 mg/kg bw/day treated males were within the range of that seen in controls and were well within the historical control data for rats of this strain and age and used in this type of studies, the differences in mean T4 value was a fortuitous finding and not related to treatment.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. Motor activity was considered similar between treated and control groups. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, lower prostate gland weights (absolute and relative to body weight) were noted for males (-3%, -5% and -21% (absolute) and -2%, -4% and -17% (relative to body weights) compared to controls for males dosed at 50, 150 and 500 mg/kg bw/day, respectively. Since no histopathological findings were noted and no effects on the reproductive capabilities of these males were apparent (see summary section 7.8), the change in prostate weights was considered to be non-adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations. For several treated females, a pale discoloured thyroid gland was observed. The incidence of this observation was equally distributed over the treated groups, i.e. 2/10, 3/10 and 2/10 in the 50, 150 and 500 mg/kg bw/day treated females respectively. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Foamy macrophage foci in the mesenteric lymph nodes were present at 150 mg/kg bw/day in females at minimal degree and at 500 mg/kg bw/day in males at minimal degree and in females up to moderate degree and multifocal necrosis was present at 500 mg/kg bw/day in a single female at moderate degree.
There were no other test item-related histologic changes.

Other effects:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment. Reproduction and develpmental toxicity parameters are included in sections 7.8.1 and 7.8.2.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects seen at highest dose tested (500 mg/kg bw/day)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: lymphatic system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results dose range finder study:

Treatment related clinical signs were observed in the females treated at 500 as well as at 1000 mg/kg bw/day. Slightly reduced food consumption was only observed after treatment at 1000 mg/kg bw/day. On the other hand, it could not be ruled out from the results obtained in this dose range finder that the macroscopic observation of a small liver and enlarged spleen in a single female at 500 mg/kg bw/day were treatment related. Therefore it was decided to extend the dose range finder with an additional group to be treated at 750 mg/kg bw/day, but for a considerable longer time period of 25 days instead of ten days for the first two groups. The results of the additional group at 750 mg/kg bw/day indicated that the reduced food consumption observed at 1000 mg/kg bw/day should be regarded as a clearly treatment-related effect and that the changes in organ sizes in 1/3 female treated at 500 mg/kg bw/day were likely a fortuitous finding and not treatment-related.

Dose formulation analysis:

The concentrations analyzed in the formulations of the groups exposed to the test item were in agreement with the target concentrations (i.e. mean accuracies between 107.8% and 110.6%). The formulations of the low and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 1.0%). No test item was detected in the control group formulation (<LLOQ). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 8 days (i.e. relative difference ≤ 3.6%). In addition, the test item was stable in water over a storage period of at least 79 days at a temperature ≤ -70°C.

Conclusions:

Based on the results of a 28 day repeated dose study with screening for reproduction and developmental effects, the parental NOAEL was found to be at least 150 mg/kg bw/day based on adverse effects on mesenteric lymph nodes (seen in 1/10 females) at 500 mg/kg bw/day.

Executive summary:

A combined 28 day repeated dose study with screening for reproductive and developmental effects was performed according to OECD/EC guidelines and GLP principles. Sodium Cocopropylenediamine Propionate was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 60-70 days). Females that failed to deliver pups were treated for 40-42 days. In females at 150 mg/kg bw/day and in both females and males at 500 mg/kg bw/day, test item-related microscopic findings were observed in the mesenteric lymph node. These findings consisted of foamy macrophage foci at 150 mg/kg bw/day in females and at 500 mg/kg bw/day in males and females which were considered non-adverse, and of multifocal necrosis in a single female at 500 mg/kg bw/day which was considered adverse.

At 500 mg/kg bw/day, an increased neutrophils-to-lymphocytes ratio was observed in both male and female rats, i.e. an approximate twice as high ratio in males as well as in females at 500 mg/kg bw/day when compared to controls. Since an increased neutrophils-to-lymphocytes ratio is considered a marker for (systemic) inflammation, a relation of the changes in these ratios to the findings in the mesenteric lymph nodes could not be ruled out, but could not be established in the current study. In males at 500 mg/kg bw/day, decreased weight of the prostate glands was found (0.79x control weight). Since no histopathological findings were noted and no effects on the reproductive capabilities of these males were apparent, the change in prostate weights was considered to be non-adverse.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, body weight, food consumption, coagulation and clinical biochemistry parameters and macroscopic findings in both sexes and T4 thyroid hormone levels in males).

Based on the adverse effects on mesenteric lymph nodes (seen in 1/10 females) at 500 mg/ kg bw/ day, a No Observed Adverse Effect Level (NOAEL) for Sodium cocopropylenediamine propionate of 150 mg/kg bw/ day was established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is a high quality guideline OECD422 study which is Klimisch 1.

System:

gastrointestinal tract

Organ:

mesenteric lymph node

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt manufactured as a ca. 30% solution in water. Inhalation is not an expected route of exposure for the product as it is handed as a liquid and Ethanol, 2, 2-iminobis-N-tallow alkyl derivatives, N-oxides is not volatile. As ECHA guidelines allows the calculation of inhalation DNELs from the oral repeat dose NOAEL where it assumes 50% absorption orally and 100% by inhalation, this conservative approach should ensure a sufficiently conservative DNEL to be calculated to protect against any incident exposure to aerosols containing β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt. It is therefore not scientifically justified to conduct and additional inhalation repeat exposure study in animals.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt manufactured as a ca. 30% solution in water. Inhalation is not an expected route of exposure for the product as it is handed as a liquid and Ethanol, 2, 2-iminobis-N-tallow alkyl derivatives, N-oxides is not volatile. The test substance is not irritating to skin but is irritating to eyes, the only local effect by inhalation could be local irritation, but this is unlikely as inhalation is not an expected route of exposure.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

A modern fully GLP compliant OECD422 oral study is available. ECHA guidelines allow the calculation of dermal DNELS based on the oral NOAEL, which is a conservative approach as oral absorption is expected to be greater than dermal due to it high water solubility. Therefore, a Dermal repeat dose study in not justified. This avoids the unnecessary use of additional animals.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

The test substance is not irritating to skin but is irritating to eyes, the only local effect by skin contact might be some mild local irritation. The long term Dermal DNELs based on the Oral NOAEL are expected to be protective from any local irritant effects. Therefore, a Dermal repeat dose study to investigate local effects in not justified. This avoids the unnecessary use of additional animals.

Mode of Action Analysis / Human Relevance Framework

In the OECD 422 study on β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt adverse effects were seen at 500 mg/kg bodyweight, of multi focal necrosis in the mesenteric lymph nodes in one female. This was accompanied by an increased neutrophils-to-lymphocytes ratio in both males and females at this top dose level, which is considered a marker for (systemic) inflammation. The NOAEL for these effects was 150mg/kg bodyweight. The mode of action would appear to be an inflammatory response to the test substance in the mesenteric lymph nodes. In most cases this resulted in foamy macrophages in the mesenteric lymph node but a more severe focal necrosis was seen in only one female at the 500mg/kg bodyweight top dose.

Justification for classification or non-classification

The adverse effects seen in the OECD 422 study on β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium on the mesenteric lymph node in one female in the 500mg/kg bodyweight top dose, is not considered to be a severe systemic toxic effect. The classification of Specific Target Organ Toxicity for a study of 28 day duration is 300mg/kg for category 2 with the cut off for category 1 of 30 mg/kg bodyweight. There were no adverse effects at 150 mg/kg bodyweight so clearly classification as STOT category 1 is not appropriate. The adverse effect seen at 500 mg/kg was of low severity only affecting one female, so this with the clear no effect level of 150mg/kg bodyweight does not justify a category 2 STOT classification. Therefore β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt does not require a classification of Specific Target Organ Toxicity.