Fatty acids, C10-12, esters with polylactic acid, sodium salts

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

other: Expert statement

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: refer to 'Remarks'

Remarks:

According to Annex XI, Item 1.2, of Regulation (EC) No. 1907/2006 (REACH), testing is not necessary if sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion. The Weight-of-Evidence assessment accounts for all relevant data from (i) the public domain, (ii) information derived from the toxicokinetic assessment of the registered substance, (iii) hazard data on the metabolites of the registered substance, and (iv) results of the repeated dose toxicity study with the registered substance. The influence of unidentified minor components of the registered substance cannot be assessed. This is considered acceptable because such components are contained in amounts below 1% or below 0.1% for known dangerous substances, such as CMR substances and are not expected to have an impact on the outcome of an acute oral toxicity study with the registered substance.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jul - 15 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Hsd: Sprague Dawley SD

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice as it is generally accepted by regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 27 - 29 days
- Weight at study initiation: 75 - 100 g
- Housing: Up to 5 animals per sex in one cage (clear polysulfone solid bottomed), nesting material was provided inside suitable bedding bags; nesting material was changed at least twice a week.
- Diet: 4 RF 21 (supplier: Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: In water bottles, ad libitum
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY: There was no indication that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analysis of water, diet and bedding material are kept on file at the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: To: 28 Jul - 15 Sep 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared weekly by suspending the appropriate amount of the test material in the vehicle (corn oil) at 40 °C under magnetic stirring for at least 16 h yielding the final concentrations of 25, 75 and 187.5 mg/mL. Each day, approx. 16 h prior to administration, the dosing formulations were again heated to 40 °C under magnetic stirring.

VEHICLE
- Justification for use and choice of vehicle: In a preliminary non-GLP toxicity test (ERBC study no. E0475) irritating effects in the stomach of rats were observed. Corn oil is known to alleviate local irritating effects.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The preparation procedure for the test item was checked in the range from 10 - 250 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A3808. Final results for all concentration levels were within the acceptability limits for concentration (80 - 120%) and homogeneity (Coefficient of Variation, CV < 10%). Samples of the dosing formulations prepared on Weeks 1 and 4 were analysed to verify the homogeneity and concentration. Results of the analyses were within the acceptability limits for suspensions as given above.

Duration of treatment / exposure:

4 weeks and 2 weeks post-exposure recovery period (satellite control and high-dose groups)

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5 (main study) and 5 (satellite control and high-dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 100, 300 and 1000 mg/kg bw/day for 28 days (ERBC study no. E0475). Mortality and adverse effects (hunched posture, swollen abdomen, rales, dyspnoea, pallor, decreased activity, distended gastrointestinal tract with gas in stomach and/or jejunum and/or ileum and/or caecum and/or colon) were observed at 1000 mg/kg bw/day. Therefore, 100, 300 and 750 mg/kg bw/day were selected as the dose levels for the main study.
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: Observation whether effects, e.g. as those observed in the dose-range finding study, are recovered.
- Post-exposure recovery period in satellite groups: 2 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during the study, each animal was observed for clinical signs. Animals were checked twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week from the start of treatment.
- Clinical observations checked: Open arena observations, changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and prior to necropsy.

FOOD CONSUMPTION:
- Time schedule: At weekly intervals, starting from Day 1 of dosing.
- Food consumption for: Each cage of rats, group mean daily intake per rat was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During the last week of treatment and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: All main phase and all recovery phase animals.
- Parameters checked: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, and Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During the last week of treatment and at the end of the recovery period.
- Animals fasted: No
- How many animals: All main phase and all recovery phase animals.
- Parameters checked: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, and Chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for general neurobehavioural examinations: Once before commencement of treatment and at least once per week from the start of treatment.
- Time schedule for sensory activiy, grip strength and motor activity: Once during Week 4 of treatment and once during Week 2 of recovery.
- Dose groups that were examined: All animals.
- General neurobehavioural examinations: Changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern).
- Battery of functions tested: Sensory activity (using auditory, visual and proprioceptive stimuli), grip strength, motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, detailed post mortem examination including examination of the external surface and orifices.
- Organs weight: Adrenal glands, Brain (cerebrum, cerebellum, medulla/pons), Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus (where present), Thyroid gland, and Uterus with cervix

HISTOPATHOLOGY: Yes
- Organs/tissues examined: Adrenal glands, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes (cervical and mesenteric), Mammary area (males and females), Ovaries, Oviducts, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal column, Spinal cord, Spleen, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus with cervix; and Vagina

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation occurred in male animals of all dose groups and in female animals of the high-dose group during treatment, non-adverse. No clinical signs were observed during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal of the high-dose group was found dead on Day 9. No clinical signs were observed prior to death. The cause of death of this animal could not be determined.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: Statistically significantly lower erythrocytes (-5%) in males after dosing phase, non-adverse. No treatment-related changes were observed after recovery phase.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: Statistically significant decrease of alanine aminotransferase (-27%) in males after dosing phase, considered not treatment-related. No changes were observed after recovery phase.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related changes were found at the weekly clinical examination, including the evaluation of neurotoxicity, functional tests and motor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

750 mg/kg bw/day: Abnormal size of the stomach (changes in non-glandular region) of most males and all females after treatment phase, adverse for rats but not relevant for humans. This observation corresponded microscopically to epithelial hyperplasia. At the end of the recovery period, no treatment-related changes remained.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

750 and 300 mg/kg bw/day: Changes in the non-glandular region of the stomach (forestomach) in all animals of the high-dose group of both sexes and in one mid-dose male (1/10) and female (1/10). Finding in the nonglandular region of the stomach consisted in epithelial hyperplasia, moderate to marked in high-dose males and mild to moderate in high-dose females; minimal in one mid-dose male and female, adverse for rats but not relevant for humans.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

MORTALITY
One male of the high-dose group (750 mg/kg bw/day) was found dead on Day 9 of dosing. Although treatment-related changes were observed in the stomach of this animals (epithelial hyperplasia), the cause of death of this animal could not be determined.

CLINICAL SIGNS
Salivation was observed in the males from all treated groups: 1/5 at 100 mg/kg bw/day and all (5/5) at 300 and 750 mg/kg/day). Salivation was also observed in all (5/5) females dosed at 750 mg/kg bw/day. No clinical signs were observed during the recovery period. No treatment-related changes were found at the weekly clinical examination, including the evaluation of neurotoxicity, functional tests and motor activity.

BODY WEIGHTS AND FOOD CONSUMPTION
No relevant changes were noted in body weight, body weight gain and food consumption in male and female animals of any group during the treatment and recovery periods.

HEMATOLOGY
After the dosing phase, erythrocyte counts were statistically significantly lower than controls in males of the high-dose group (750 mg/kg bw/day). Due to the minimal severity (5%) and the absence of changes in any other related parameters (e.g. haemoglobin and haematocrit), this finding was not considered to be adverse. Following the two week recovery periode, statistically significant differences of monocytes between control and treated males was recorded. This finding was not observed at the end of treatment, therefore, was considered incidental.

CLINICAL CHEMISTRY
A statistically significant decrease of alanine aminotransferase (-27%) recorded in males of the mid-dose group (300 mg/kg bw/day) was not considered treatment-related due to the lack of a dose-dependence. No changes were observed following the two weeks recovery period.

ORGAN WEIGHTS
Organ weight variations were in the range of expected spontaneous changes in rats of the same age and considered unrelated to treatment. There were no treatment-related changes in organ weights at the end of the recovery period.

MACROSCOPIC OBSERVATIONS
Following treatment with the test substance, treatment-related macroscopic changes in the stomach (abnormal size) of most high-dose males and all females (750 mg/kg bw/day) were observed. This observation corresponded microscopically to epithelial hyperplasia. Any other observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age and were considered incidental and unrelated to treatment. No changes were recoreded after the recovery period.

MICROSCOPIC OBSERVATIONS
After the treatment phase, changes considered to be associated with treatment were present in the stomach of animals of both sexes of the high- and mid-dose groups (dosed at 300 and 750 mg/kg bw/day). Treatment-related changes were seen in the non-glandular region of the stomach (forestomach) in all animals (10/10) of the high-dose group of both sexes and in one mid-dose male (1/10) and female (1/10). The finding of the non-glandular region of the stomach consisted in epithelial hyperplasia, moderate to marked in high-dose males and mild to moderate in high-dose females; minimal in one mid-dose male and female. Epithelial hyperplasia was characterized by uniform thickening of all layers of the epithelium and submucosal chronic inflammation and oedema. In some high-dose treated males, the epithelial hyperplasia was associated with erosion / ulceration of squamous epithelium. These findings were considered treatment-related and potentially reflected an irritant effect of the test substance. Any microscopic observation other than those mentioned above were within the range of occasionally observed and expected spontaneous changes in rats of the same age and, therefore, considered incidental and unrelated to treatment.
After two weeks of recovery, histopathological changes were observed in the non-glandular region of the stomach (forestomach) in all high-dose male animals from minimal to moderate degree; the lower severity and multifocal distribution when compared to the male rats dosed at 750 mg/kg bw/day following the dosing phase indicated a partial recovery. There were no microscopic observations in the non-glandular region of the stomach of high-dose females at the end of the recovery period, indicating full recovery of gastric findings in females. In light of the above findings, in high- and mid-dose animals of both sexes, the treatment-related epithelial hyperplasia of the non-glandular stomach was not considered to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Detailed data are provided in tabular form under 'Attached background material'.

Conclusions:

Histopathological changes observed in the non-glandular region of the stomach of animals treated at 300 and 750 mg/kg bw/day were considered to be due to local irritant effects of the test substance and were not sufficiently severe to become adverse and not relevant for humans due to their location. The No-Observed-Adverse-Effect-Level (NOAEL) was, therefore, determined to be 750 mg/kg bw/day, the highest dose tested.

Data source

Reference

Reference Type:

other: Expert Statement

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The acute oral toxicity was assessed from the available data on single constituents, related substances and on the metabolites of enzymatic ester hydrolysis as well as results of the short-term repeated dose toxicity study with the registered substance.

GLP compliance:

no

Test type:

other: Weight-of-Evidence assessment that compiles various tests on metabolism and acute oral toxicity on related substances and metabolites

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C10-12, esters with polylactic acid, sodium salts

EC Number:

700-937-1

Cas Number:

1312021-45-6

Molecular formula:

not available

IUPAC Name:

Fatty acids, C10-12, esters with polylactic acid, sodium salts

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

other: data on several rat strains are used

Sex:

male/female

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

other: Studies using various vehicles were assessed

Doses:

Depending on the substance and duration of the studies, the maximum doses ranged between 2400 and 6000 mg/kg bw. Details are given in the expert statement.

No. of animals per sex per dose:

variable

Control animals:

other: in some studies control animals are included

Results and discussion

Preliminary study:

Based on bibliographical knowledge and on current valid ADI (acceptable daily intake) of 20 mg/kg bw/day for sodium stearoyl lactylate which is approved to be used in food and feed, the oral LD50 is predicted to be higher than 2000 mg/kg bw. No mortality and no findings of necropsy are expected.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

4 880 mg/kg bw

Based on:

other: calculated by conservative mathematical approach

Remarks on result:

other: weight of evidence

Mortality:

None expected

Clinical signs:

other: No unusual signs predicted

Gross pathology:

No effects on gross pathology are predicted

Other findings:

None

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

Result by weight of evidence: The calculated LD50 (oral, rat) : 4.88g/kg bw

Executive summary:

The registered substance was assessed for its acute oral toxicity by means of a weight-of-evidence approach. The oral LD50 values of structural similar compounds exceed 5 g/kg bw. Additional long-term toxicity data of the lactylic ester of stearic acid lead to an acceptable daily intake (ADI) of 20 mg/kg bw/day for that substance. Moreover, lactylic esters of fatty acids may be safely used in food per 21 CFR 172.848. Lactylic acid esters of long and short chain fatty acids are metabolised in the same way, hence it can be assumed that the registered substance shows no long-term toxicity potential. This is further supported by the results of the short-term repeated dose toxicity study with the registered substance. The impurity sodium lactate, which the registered substance contains up to approx. 10%, has an ADI that is not limited. Hence, a negative effect on the oral LD50 can be ruled out.

In summary, the acute oral toxicity of the registered substance is assumed to be very low. The presented data from several independent sources of information are considered sufficient to evaluate the acute oral toxicity of the registered substance. According to Annex XI, Item 1.2, of Regulation (EC) No. 1907/2006 (REACH), further testing on vertebrate animals shall be omitted if sufficient weight of evidence for the presence or absence of a particular dangerous property is available.