Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 24, 2017 to December 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Deviations did not affect the outcome of the results of this study.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Deviations did not affect the outcome of the results of this study.

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
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. 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal Identification: At study assignment, each animal was identified using an earmark and tattoo.
- Environmental Acclimation: The animals were allowed to acclimate to the test facility toxicology accommodation for 6 d before the commencement of dosing.
- Selection, Assignment, Replacement, and Disposition of Animals: Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

- Housing: On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. The room(s) in which the animals were kept was documented in the study records. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water. Each cage was clearly labeled with a color-coded cage card indicating test facility study no., group, animal number(s), and sex.

- Environmental conditions: Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 22°C with an actual daily mean relative humidity of 40 to 59%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

- Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 h. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the test facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to food for a maximum of 2 h. Periodic analysis of the water is performed, and results of these analyses are on file at the test facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is the intended route of human exposure.

Vehicle:

water

Details on oral exposure:

Preparation of test formulation:

Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. A correction factor was used to correct for the composition of the test substance (53.2% in water). The dosing formulations were prepared daily as a suspension and dosed within 5 h after preparation of the formulation. Test substance dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection and analysis: Stability samples were kept at room temperature under normal laboratory light conditions for 5 h, and then placed on dry ice. All other samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to ABL BV (within 1 week of preparation). The analytical laboratory was notified before shipment of the samples. Upon receipt at the analytical laboratory, the samples were stored in the freezer ≤ -70 °C until analysis. Residual samples were discarded after completion of the sample analysis.

Dose formulation sample collection schedule

Interval Concentration Homogenecity Stability
Week 1 all groups Group 2 and 4 Group 2 and 4
Week 6 all groups Group 2 and 4 N/A
Week 13 all groups Group 2 and 4 N/A

Analytical method: Analyses were performed using a validated analytical procedure using LC-MS/MS.

Duration of treatment / exposure:

90 d

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels are based on results of a 14 d dose range finding study withthe test substance.
- Rationale for animal assignment: random, by computer-generated algorithm.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning during the first administration of the test substance and lasting throughout the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during Pretreatment in all Main Study (including spare animals), and at the end of the Dosing Period in Week 13 in all group 1 (control) and 4 (1000 mg/kg bw) Main study animals. Since the above ophthalmic examinations did not reveal treatment-related effects, examination was not performed on the animals of Groups 2 (100 mg/kg bw) and 3 (300 mg/kg bw).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals: all animals
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12-13.
- Dose groups that were examined: 5 animals per sex per group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Hearing ability, pupillary reflex, static righting reflex and locomotor activity

HEMATOLOGY: White blood cells (WBC) Red Blood Cell Distribution Width (RDW), neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular, haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).

COAGULATION: Blood samples were processed for plasma, and plasma was analyzed for Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Bile Acids, Chloride, Total Bilirubin, Calcium, Urea Inorganic Phosphate (Inorg. Phos)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals sacrificed on Day 92, 93
HISTOPATHOLOGY: Yes

Other examinations:

No

Statistics:

All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 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 2 observations.
The following pairwise comparisons were made:
Group 2 (100 mg/kg bw) vs. Group 1 (control)
Group 3 (300 mg/kg bw) vs. Group 1 (control)
Group 4 (1000 mg/kg bw) vs. Group 1 (control)

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
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.

Incidence
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, non-treatment-related

Description (incidence and severity):

No toxicological relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Salivation seen after dosing among all males and females treated at 1000 mg/kg was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to the test substance rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period 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:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period. The incidental statistically significant changes in body weight gain of females at 100 mg/kg were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the study period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment. The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test substance.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Lower neutrophil counts (males only) and lower haemoglobin and haematocrit levels, mean corpuscular volume and/or mean corpuscular haemoglobin levels were noted in both sexes at 1000 mg/kg. Decreased red blood cell counts and increased platelet counts were statistically significant in females at the highest dose of 1000 mg/kg. These alterations in hematological parameters were unrelated to administration of the test item due to the minimal magnitude of the change and absence of a clear dose response, and absence of corroborative findings in the opposite sex. Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In females, higher total protein and calcium levels were noted at 1000 mg/kg and higher albumin at 300 and 1000 mg/kg and lower inorganic phosphate was noted in females at 100, 300 and 1000 mg/kg. In males at 100 and 1000 mg/kg single animals showed high level of aspartate aminotransferase activity (ASAT), at the incidence observed this was considered not to be toxicologically relevant. Any other values in treated males and females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weights were recorded at 1000 mg/kg in males (relative to body weights) and in females (absolute and relative to body weights). There was no microscopic correlation with the increase in liver weights. There were no other test substance-related organ weight changes

Gross pathological findings:

no effects observed

Description (incidence and severity):

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance related microscopic findings were noted in the kidneys (vacuolation of tubular epithelium) of males at 1000 mg/kg and in females at 300 and 1000 mg/kg and in the urinary bladder (vacuolation of the urothelium) of females at 1000 mg/kg. These findings were correlated to increased kidney weights in males at 1000 mg/kg up to 15% and in females at 300 and 1000 mg/kg up to 28%, however no other additional morphologic alteration were noted that suggest any organ dysfunction. Therefore, the vacuolation in kidneys and urinary bladder was considered to be a non-adverse microscopic alteration.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

Dose formulation analyses:
No test substance was detected in the Group 1 formulation. The concentrations analysed in the formulations of Group 2, 3 and 4 (week 1, 6 and 13) were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 prepared 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 5 h (i.e. relative difference ≤10%). In addition the test substance was stable in Elix water over a storage period of at least 58 d at a temperature ≤ -70°C.

Mortality: No mortality occurred during the study period.

Clinical Observations: No toxicological relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation seen after dosing among all males and females treated at 1000 mg/kg bw was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to the test substance rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period 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.

Body Weights and Body Weight Gains: Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
The incidental statistically significant changes in body weight gain of females at 100 mg/kg bw were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food Consumption: Food consumption before or after correction for body weight was similar to the control level over the study period.

Ophthalmic Examinations: No ophthalmology findings were noted that were considered to be related to treatment. The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test substance.

Functional Observations: Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. The statistically significant change in hind limb grip strength of females at 300 mg/kg bw was considered to be unrelated to treatment since no trend was apparent regarding dose.

Hematology: Lower neutrophil counts (males only) and lower haemoglobin and haematocrit levels, mean corpuscular volume and/or mean corpuscular haemoglobin levels were noted in both sexes at 1000 mg/kg bw. Decreased red blood cell counts and increased platelet counts were statistically significant in
females at the highest dose of 1000 mg/kg bw. These alterations in hematological parameters were unrelated to administration of the test substance due to the minimal magnitude of the change and absence of a clear dose response, and absence of corroborative findings in the opposite sex. Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Coagulation: Coagulation parameters of treated rats were considered not to have been affected by treatment. The statistically significant lower prothrombin time (PT) of females at 100 and 300 mg/kg bw was not considered to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical Chemistry: In females, higher total protein and calcium levels were noted at 1000 mg/kg bw and higher albumin at 300 and 1000 mg/kg bw and lower inorganic phosphate was noted in females at 100, 300 and 1000 mg/kg bw. In males at 100 and 1000 mg/kg bw single animals showed high level of aspartate aminotransferase activity (ASAT), at the incidence observed this was considered not to be toxicologically relevant. Any other values in treated males and females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance.

Gross Pathology: There were no test substance-related gross observations. All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this age and strain.

Organ Weights: Statistically significant test substance-related higher kidney weights (absolute and relative to body weight) were recorded in females of all test substance treated dose groups and in males at 1000 mg/kg bw. The microscopic correlate for this kidney weight increase was considered tubular vacuolation, Higher liver weights were recorded at 1000 mg/kg bw in males (relative to body weights) and in females (absolute and relative to body weights). There was no microscopic correlate for the increase in liver weights. There were no other test substance related organ weight changes.

Histopathology: Test substance related microscopic findings were noted in the kidneys of males at 1000 mg/kg bw and in females at 300 and 1000 mg/kg bw and in the urinary bladder of females at 1000 mg/kg bw.

Kidneys: Tubular vacuolation was recorded in males at 1000 mg/kg bw (minimal-slight) and in females at 300 mg/kg bw (minimal-slight) and 1000 mg/kg bw (minimal-moderate). This finding correlated with the increased kidney weights recorded at necropsy (males at 1000 mg/kg bw and females at 300 and 1000 mg/kg bw). The minimal tubular vacuolation recorded in a single female of the control group and two females at 100 mg/kg bw was considered to be within background pathology for rats of this age and strain.

Urinary bladder: An increased incidence and severity of urothelial vacuolation was recorded in a few females at 1000 mg/kg bw (minimal-slight). The minimal vacuolation of the urothelium recorded in a single female of the control group was considered to be within background pathology for rats of this age and strain.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

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 adverse effects observed up to the highest tested dose

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the NOAEL of the test substance for repeated dose toxicity in rats was determined to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance, according to Guideline OECD 408, in compliance with GLP. 10 male and 10 female Wistar rats per group were treated with test substance for 13 weeks (90 d) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. Doses were finalised based on the range finding study. Test formulations were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations prepared were considered stable, for at least 5 h at room temperature. At histopathological examination an increased incidence and severity of vacuolation of the tubular epithelium of the kidneys was recorded in males at 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day. In addition vacuolation of the urothelium of the urinary bladder was seen in females at 1000 mg/kg bw. These findings were correlated to increased kidney weights in males at 1000 mg/kg bw up to 15% and in females at 300 and 1000 mg/kg bw up to 28%, however no other additional morphologic alteration were noted that suggest any organ dysfunction. Therefore the vacuolation in kidneys and urinary bladder was considered to be a non-adverse microscopic alteration (Kerlin et al., 2016; Palazzi et al., 2016). Slight increase in total protein, albumin and calcium levels and decrease in inorganic phosphate levels were noted in treated females predominantly at 1000 mg/kg bw/day. These findings were only slight with no histopathological correlation and therefore no toxicological relevance was ascribed to these changes. Liver weight increase was recorded for males and females at 1000 mg/kg bw/day and kidney weight increase was recorded for females at 100 mg/kg bw/day. Although a clear weight increase was noted in liver (maximum absolute increase 24%) and kidney (maximum absolute increase 17%), these changes occurred with no evidence of organ dysfunction, no toxicologically relevant clinical biochemical findings and with no histopathological correlation. Therefore these effects were considered to be non-adverse (Hall et al., 2012; Kerlin et al., 2016; Palazzi et al., 2016). Slight changes in haematological parameters were noted as lower neutrophil counts (males only) and lower haemoglobin and haematocrit levels, mean corpuscular volume or mean corpuscular haemoglobin levels in both sexes. These changes were slight and were found without a histopathological correlation and therefore considered not adverse. No other toxicologically significant changes were noted in this study. Therefore, under the study conditions, the NOAEL of the test substance for repeated dose toxicity in rats was determined to be 1000 mg/kg bw/day (Beerens-Heijnen, 2018).

References:

1) Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J. (2016) Scientific and Regulatory Policy Committee: Recommended ("Best") Practices for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies. Toxicol. Pathol. 44(2), 147 -62.

2) Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gröters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M (2016). Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicol. Pathol. 44(6), 810-24.

3) Hall, A. P., Elcombe, C. R., Foster, J. R., Harada, T., Kaufmann, W., Knippel, A., Kuttler, K., Malarkey, D. E., Maronpot, R. R., Nishikawa, A., Nolte, T., Schulte, A., Strauss, V., and York, M. J. (2012). Liver hypertrophy: A review of adaptive (adverse and non-adverse) changes—Conclusions from the 3rd International ESTP Expert Workshop. Toxicol Pathol 40, 971–94.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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 study was conducted to determine the repeated dose toxicity of the test substance, according to Guideline OECD 408, in compliance with GLP.10 male and 10 female Wistar rats per groupwere treated with test substance for 13 weeks (90 d) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day.Doses were finalised based on the range finding study.Test formulations were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Test formulations prepared were considered stable, for at least 5 h at room temperature.At histopathological examination an increased incidence and severity of vacuolation of the tubular epithelium of the kidneys was recorded in males at 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day. In addition vacuolation of the urothelium of the urinary bladder was seen in females at 1000 mg/kg bw. These findings were correlated to increased kidney weights in males at 1000 mg/kg bw up to 15% and in females at 300 and 1000 mg/kg bw up to 28%, however no other additional morphologic alteration were noted that suggest any organ dysfunction. Therefore the vacuolation in kidneys and urinary bladder was considered to be a non-adverse microscopic alteration (Kerlin et al., 2016; Palazzi et al., 2016).Slight increase in total protein, albumin and calcium levels and decrease in inorganic phosphate levels were noted in treated females predominantly at 1000 mg/kg bw/day. These findings were only slight with no histopathological correlation and therefore no toxicological relevance was ascribed to these changes.Liver weight increase was recorded for males and females at 1000 mg/kg bw/day and kidney weight increase was recorded for females at 100 mg/kg bw/day. Although a clear weight increase was noted in liver (maximum absolute increase 24%) and kidney (maximum absolute increase 17%), these changes occurred with no evidence of organ dysfunction, no toxicologically relevant clinical biochemical findings and with no histopathological correlation. Therefore these effects were considered to be non-adverse (Hall et al., 2012; Kerlin et al., 2016; Palazzi et al., 2016).Slight changes in haematological parameters were noted as lower neutrophil counts (males only) and lower haemoglobin and haematocrit levels, mean corpuscular volume or mean corpuscular haemoglobin levels in both sexes. These changes were slight and were found without a histopathological correlation and therefore considered not adverse. No other toxicologically significant changes were noted in this study.Therefore, under the study conditions, the NOAEL of the test substance for repeated dose toxicity in rats was determined to be 1000 mg/kg bw/day (Beerens-Heijnen, 2018).

References:

1) Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J. (2016) Scientific and Regulatory Policy Committee: Recommended ("Best") Practices for Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies. Toxicol. Pathol. 44(2), 147 -62.

2) Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gröters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M (2016). Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop. Toxicol. Pathol. 44(6), 810-24.

3) Hall, A. P., Elcombe, C. R., Foster, J. R., Harada, T., Kaufmann, W., Knippel, A., Kuttler, K., Malarkey, D. E., Maronpot, R. R., Nishikawa, A., Nolte, T., Schulte, A., Strauss, V., and York, M. J. (2012). Liver hypertrophy: A review of adaptive (adverse and non-adverse) changes—Conclusions from the 3rd International ESTP Expert Workshop. Toxicol Pathol 40, 971–94.

Justification for classification or non-classification

Based on the results of the subchronic repeated dose toxicity study in rats, the test substance does not warrant any classification according to EU CLP (1272/2008) criteria.