fatty acid chloride, C12, reaction product with sodium glutamate and sodium hydroxide

Endpoint summary

Description of key information

The subacute toxicity of the registration substance "sodium N-lauroyl glutamte" was evaluated based on the read-across approach using the repeated dose toxicity data for sodium N-cocoyl glycinate.

The registration substance and the read-across sources are amides of fatty acids and amino acids and can be characterized as "N-fatty acyl amino acids", of which endogeous occurence and metabolism are known. Based on the comparable chemical structure, comparable phys-chem data and expected comparable metabolism, these compound are likely exhibit comparable toxicity profiles.

The repeated dose toxicity of sodium N-cocoyl glycinate was investigated according to the Guideline OECD 407. The test material, composed of 70% sodium cocoyl glycinate and 20 % sodium chloride, was dissolved in water and was administered to rats via gavage at dosages of 0, 62.5, 250, and 1000 mg/kg bw/day, corresponding to up to 700 mg/kg bw/day of sodium cocoyl glycinate. The study included animals for recovery groups for control and high dose and these animals were allowed to recover for 14 days.

Up to the highest dose no clinical signs, no effect on body weight, no effect on food consuption, no effect on FOB, no effect on hematology, no effect on clinical biochemistry, no effect on macroscopic observations, no effect on organ weight were found. Upon histopathological investigation, lesions in forstomach was found for animals of high dose, which consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. The effect in the forstomach was completely reversible and considered as irritative local effect.

The systemic NOAEL of 1000 mg/kg bw/day, corresponding to 700 mg/kg bw/day of sodium N-cocoyl glycinate, was obtained.

Likewise the registration substance "sodium N-lauroyl glutamate" is likely of low aubacute toxicity. Taking account the molecular weight difference of registration substance and the read-across supporting substance (373 vs. 279 g/mol) the systemic NOAEL of 940 mg/kg bw/day can be obtained for the registration substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Justification for read-across is provided in chapter 13 as separate statement

Reason / purpose for cross-reference:

assessment report

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No apparent systemic effect up to the highest dose

Key result

Critical effects observed:

no

Conclusions:

The subacute toxicity the registration substance sodium N-lauroyl glutamate is derived based on the read-across sodium N-cocoyl glycinate. A low subacute toxicity can be reliably derived. The NOAEL of 940 mg/kg bw/day is recommended for the purpose of risk assessment.

Executive summary:

The subacute toxicity of the registration substance "sodium N-lauroyl glutamte" was evaluated based on the read-across approach using the repeated dose toxicity data for sodium N-cocoyl glycinate.

The registration substance and the read-across sources are amides of fatty acids and amino acids and can be characterized as "N-fatty acyl amino acids", of which endogeous occurence and metabolism are known. Based on the comparable chemical structure, comparable phys-chem data and expected comparable metabolism, these compound are likely exhibit comparable toxicity profiles.

The repeated dose toxicity of sodium N-cocoyl glycinate was investigated according to the Guideline OECD 407. The test material, composed of 70% sodium cocoyl glycinate and 20 % sodium chloride, was dissolved in water and was administered to rats via gavage at dosages of 0, 62.5, 250, and 1000 mg/kg bw/day, corresponding to up to 700 mg/kg bw/day of sodium cocoyl glycinate. The study included animals for recovery groups for control and high dose and these animals were allowed to recover for 14 days.

Up to the highest dose no clinical signs, no effect on body weight, no effect on food consuption, no effect on FOB, no effect on hematology, no effect on clinical biochemistry, no effect on macroscopic observations, no effect on organ weight were found. Upon histopathological investigation, lesions in forstomach was found for animals of high dose, which consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. The effect in the forstomach was completely reversible and considered as irritative local effect.

The systemic NOAEL of 1000 mg/kg bw/day, corresponding to 700 mg/kg bw/day of sodium N-cocoyl glycinate, was obtained.

Likewise the registration substance "sodium N-lauroyl glutamate" is likely of low repeated dose toxicity. Taking account the molecular weight difference of registration substance and the read-across supporting substance (373 vs. 279 g/mol) the systemic NOAEL of 940 mg/kg bw/day can be obtained for the registration substance.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

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: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories B.V.,Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals:
Group 1: 10 males and 10 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 10 males and 10 females
Group 10: 1 male and 1 female
Age (at Delivery): Ca. 7 weeks
Body Weight Range
(at Acclimatization):
Males: 199 to 233 g (mean 214 g)
Females: 133 to 154 g (mean 144 g)
Identification:
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Randomly allocated to groups by body weight.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS

Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).

Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

Water: Community tap-water from Itingen was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of administration: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily.
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 62.5 mg/kg/day
Group 3: 250 mg/kg/day
Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D43306.
Dose Volume: 10 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6.25 mg/mL/day
Group 3: 25 mg/mL/day
Group 4: 100 mg/mL/day
Duration of Pre-Randomization Phase: 1 day
Duration of Acclimatization Phase: At least 5 days
Duration of Treatment Phase: 28 days
Duration of Recovery Phase: 14 days

PREPARATION OF DOSING SOLUTIONS:
-For the purpose of this study the test material was prepared at the appropriate concentrations
as a solution in highly purified water.

VEHICLE
-Highly purified water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days).

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The test item was used as analytical standard.

Dose formulation samples (primary samples or duplicates) were discarded upon written confirmation by the study director after acceptance of the draft report.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

two groups; one for the necropsy on one day after the treatment and one for the necropsy on 14 days after treatment.

No. of animals per sex per dose:

Group 1: 10 males and 10 females at 0 mg/kg bw/day
Group 2: 5 males and 5 females at 62.5 mg/kg bw/day
Group 3: 5 males and 5 females at 250 mg/kg bw/day
Group 4: 10 males and 10 females at 1000 mg/kg bw/day
Group 10: 1 male and 1 female (reserve animals)

Reserve animals were exchanged as needed during the acclimatization period and then removed from the study. Any raw data collected during the acclimatization period on reserve animals were not reported but retained in the raw data.

Control animals:

yes, concurrent vehicle

Details on study design:

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily.
Daily Target Dose Level:
Group 1: 0 mg/kg/day
Group 2: 62.5 mg/kg/day
Group 3: 250 mg/kg/day
Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D43306.
Dose Volume: 10 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6.25 mg/mL/day
Group 3: 25 mg/mL/day
Group 4: 100 mg/mL/day
Duration of Pre-Randomization Phase: 1 day
Duration of Acclimatization Phase: At least 5 days
Duration of Treatment Phase: 28 days
Duration of Recovery Phase: 14 days

Positive control:

None

Observations and examinations performed and frequency:

-Viability / Mortality
Observations for viability / mortality were recorded twice daily.

-Daily Observations
The animals were observed daily for clinical signs before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period, and once daily during days 1 to 14 of the recovery period.

-Weekly Behavioral Observations
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

-Functional Observational Battery (Screen)
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results are present in the summary and individual tables of the weekly detailed clinical observations under week 4.

-Grip Strength
Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

-Locomotor Activity
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

-Food Consumption
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

-Body Weights
Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

-Clinical Laboratory Investigations
Blood and Urine Sampling:
After 4 Weeks: 20-Feb-2012 (allocation A and B)
After 6 Weeks: 05-Mar-2012 (allocation B)

Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.

-Hematology
The following hematology parameters were determined:

-Complete Blood Cell Count

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium, high fluorescence)
Leukocyte count, total
Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count

-Hemoglobin Derivatives

Methemoglobin Heinz bodies (slides were prepared but not evaluated)
Coagulation

Prothrombin time (= Thromboplastin time) Activated partial Thromboplastin time

-Clinical Biochemistry
The following clinical biochemistry parameters were determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Alkaline phosphatase
Bile acids Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

-Urinalysis
The following urine parameters were determined:

-Physical Examination

Urine volume (18 hour)
Specific gravity (relative density)
Color
Appearance

-Chemical Examination

pH value
Nitrite
Protein
Glucose
Ketones Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

-Necropsy
Sacrifice:

After 4 Weeks: 20-Feb-2012 (allocation A)
After 6 Weeks: 05-Mar-2012 (allocation B)

All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

A blood sample was taken from each animal by heart puncture (ca. 2 mL) into appropriate tubes (Vacutainer glass tubes containing SST-Gel and Clot Activator or the equivalent) for serum preparation and then placed on ice or cool plates. Following centrifugation, the serum was divided in 2 aliquots of at least 350 µL and transferred to plastic (polypropylene) tubes. These samples were stored at approximately -80 °C and protected from light in case they were needed for analysis of hormone activity.

-Organ Weights
The organs from allocation A and B animals were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

-Histotechnique
All organ and tissue samples, as defined under Histopathology , were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

-Histopathology
Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Liver. Lungs and stomach of animals from the low and middle dose groups were also evaluated. Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.

A description of all abnormalities is attached (see Appendix VI ). Attempts were made to correlate gross observations with microscopic findings.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

-Viability / Mortality
- Organ Weights

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

• The Dunnett-test [see References (1)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test [see References (2)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test [see References (3)].

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):

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, microscopic changes related to the treatment with the test item were observed in the forestomach of animals at 1000 mg/kg/day. These lesions consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. After the 14-day recovery period, these changes were completely reversible.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No substance related mortality and no test item-related clinical signs noted at any dose level during treatment and recovery period.

BODY WEIGHT AND WEIGHT GAIN:
No test item-related findings in mean body weights or body weight gain.

HAEMATOLOGY:
No test item-related changes in haematology at any dose level.

CLINICAL CHEMISTRY:
No test item-related changes in clinical biochemistry at any dose level.

URINALYSIS:
No test item-related changes at any dose level.

NEUROBEHAVIOUR:
No test item-related neurobehavioural signs during daily or weekly observations and no findings during the functional observational battery or related tests (grip strength, locomotor activity).

ORGAN WEIGHTS:
No test item-related findings at any dose level.

GROSS PATHOLOGY:
No test item-related findings at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Test item-related findings were restricted to microscopic changes in the forestomach of animals at the limit dose of 1000 mg/kg body weight.

HISTOPATHOLOGY: NEOPLASTIC:
No test item-related findings at any dose level.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects with regard to systemic toxicity; effect in forstomach was reversible.

Critical effects observed:

not specified

Conclusions:

The repeated dose toxicity of sodium N-cocoyl glycinate was investigated according to the Guideline OECD 407. The NOAEL of 1000 mg/kg/day of test material, corresponding to 700 mg/kg bw/day for sodium N-cocoyl glycinate was established.

Executive summary:

The repeated dose toxicity of sodium N-cocoyl glycinate was investigated according to the Guideline OECD 407. The test material, composed of 70% sodium cocoyl glycinate and 20 % sodium chloride, was dissolved in water and was administered to rats via gavage at dosages of 0, 62.5, 250, and 1000 mg/kg bw/day, corresponding to up to 700 mg/kg bw/day of sodium cocoyl glycinate. The study included animals for recovery groups at 0 and 1000 mg/kg bw/day that were allowed to recover for 14 days.

Up to the highest dose no clinical signs, no effect on body weight, no effect on food consuption, no effect on FOB, no effect on hematology, no effect on clinical biochemistry, no effect on macroscopic observations, no effect on organ weight were found. Upon histopathological investigation, lesions in forstomach was found for animals of 1000 mg/kg bw/day, which consisted of acanthosis, parakeratosis, inflammation, ulceration and erosions. The effect in the forstomach was completely reversible and considered as irritative local effect.

The NOAEL of 1000 mg/kg bw/day, corresponding to 700 mg/kg bw/day of sodium N-cocoyl glycinate, was obtained.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

940 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

one valid study on read-across substance

Justification for classification or non-classification

The subacute toxicity the registration substance sodium N-lauroyl glutamate is derived based on the read-across sodium N-cocoyl glycinate. A low subacute toxicity can be reliably derived. No classification is assigned.