Magnesium octadecylbenzenesulphonate

Repeated dose toxicity: oral

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Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2014 to 07 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

Batch No.: E00873-025-001
Physical Description: Black solid
Storage Conditions: Kept in a controlled room temperature area

Test animals

Species:

rat

Strain:

other: Wistar (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 8 weeks old).
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 229 grams; females: 167 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 26 August 2014 to 07 October 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to administration and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance. Formulations were heated to a maximum temperature of 95.5°C for a maximum duration of 75 minutes to obtain visual homogeneity. Formulations were allowed to cool down to 40°C or lower before dosing.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information from the Sponsor.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Project 505730). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 (Groups 1&4: 5 main, 5 recovery), 5 (Groups 2&3: 5 main)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 14-day dose range finding study (Project 505704).

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were conducted in all animals immediately after dosing, (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (project 505704)). Once prior to start of treatment and at weekly intervals during the treatment phase this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: on the day of necropsy at the end of the treatment and recovery phase
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of necropsy at the end of the treatment and recovery phase
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

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

Sacrifice and pathology:

GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Time schedule for examinations: after 28 days of treatment (5 animals/sex/Group) and after 14 days of recovery (5 animals/sex/Groups 1 and 4)
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period.
No clinical signs of toxicity or abnormalities during weekly arena observations were noted during the observation period. Salivation was seen intermittently after dosing among all animals at 1000 mg/kg during the treatment period only. Considering 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 taste of the test substance rather than a sign of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
At 1000 mg/kg, body weight gain was lower during the treatment period (males) and the recovery period (both sexes), achieving a level of statistical significance on most occasions. Mean body weight was also statistically significantly lower for both sexes at commencement of the recovery phase, but
mean weight gain over the recovery period compared to Day 1 of the recovery period was similar to control levels for both sexes. Body weights and body weight gain of other treated animals remained in the same range as controls over the study period.

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

HAEMATOLOGY
The following statistically significant changes in haematology parameters at the end of treatment were considered to be related to treatment at 1000 mg/kg:
- Higher white blood cell counts (WBC) in both sexes,
- Shorter activated partial thromboplastin time (APTT) in males.
At the end of the recovery period, only activated partial thromboplastin time remained lower in males. The lower relative eosinophil counts in both sexes at 1000 mg/kg at the end of treatment were considered to have occurred secondarily to the higher absolute white blood cell counts, and hence considered unrelated to a primary effect of the test substance. Other haematological parameters were considered to have been unaffected by treatment. The higher red blood cell distribution width (RDW) of females at 1000 mg/kg at the end of the recovery period was considered unrelated to treatment since this finding was absent at the end of the treatment period.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters were considered to be related to treatment:
- Higher alanine aminotransferase activity (ALAT) in males and females at 1000 mg/kg, and in males at 300 mg/kg (not statistically significant at 300 mg/kg),
- Higher aspartate aminotransferase activity (ASAT) in males and females 1000 mg/kg (males at 300 mg/kg also showed an apparent increase of this parameter),
- Higher alkaline phosphatase activity in males at 1000 mg/kg,
- Lower total protein level in males and females at 300 and 1000 mg/kg, and in males also at 100 mg/kg,
- Lower albumin level in males and females at 300 and 1000 mg/kg, and in males also at 100 mg/kg,
- Higher total bilirubin level in males and females at 1000 mg/kg, and in females also at 100 and 300 mg/kg,
- Lower creatinine level in males at 1000 mg/kg,
- Lower cholesterol level in males and females at 1000 mg/kg,
- Higher bile acid level in males and females at 300 and 1000 mg/kg (not statistically significant for males at 300 mg/kg),
- Lower calcium level in males and females at 100, 300 and 1000 mg/kg (not statistically significant for males at 100 mg/kg).
At the end of the recovery period, only total protein level remained lower in males at 1000 mg/kg. Other statistically significant changes in clinical biochemistry parameters were considered unrelated to treatment as these occurred in the absence of a dose-related trend, remained within the range considered normal for rats of this age and strain and/or were absent at the end of the treatment period.

NEUROBEHAVIOUR
Motor activity was considered to have been unaffected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. At the end of the treatment period, males showed an apparent trend towards an increase in motor activity (ambulations). Means were not statistically significant and remained within the range considered normal for rats of this age and strain. Means for total movements did not show a doserelated trend. The motor activity pattern over the 1-hour measurement period was generally similar to the variation encountered for other groups. Overall, these data were considered not to point to a treatment-related effect.

ORGAN WEIGHTS
The following changes in absolute organ weights and relative organ weights (organ to body weight ratio) at the end of the treatment period were considered to be related to treatment:
- Higher relative kidney weights in males at 1000 mg/kg,
- Lower absolute prostate and seminal vesicle weight and lower relative prostate weight in males at 1000 mg/kg,
- Lower absolute and relative heart weight in females at 1000 mg/kg,
- Lower absolute and relative thymus weight in females at 1000 mg/kg,
- Higher relative spleen weight in females at 1000 mg/kg.
At the end of the recovery period, only relative kidney weights in males at 1000 mg/kg remained slightly higher. Other statistically significant changes in organ weights and organ to body weight ratios were considered to be unrelated to treatment, since these changes occurred in the absence of a doserelated trend, remained within the range considered normal for rats of this age and strain and/or were absent at the end of the treatment period.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be unrelated to treatment with the test substance.

HISTOPATHOLOGY
Multifocal necrosis of the liver (slight) was recorded in 1/5 males at 1000 mg/kg at the end of the treatment period. After the 14-day treatment-free recovery period this finding had fully recovered. There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Accuracy of preparation: The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation = 10%).

Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:

In this OECD407 study, a No Observed Adverse Effect Level (NOAEL) for the test material of 300 mg/kg was established based on the presence of multifocal necrosis of the liver in one male at 1000 mg/kg, and the magnitude of change of primarily alanine aminotransferase activity at 1000 mg/kg, in combination with other clinical biochemistry changes that point to a disturbance in liver function.

Executive summary:

The study was conducted in accordance with OECD 407. Based on the presence of multifocal necrosis of the liver in one male at 1000 mg/kg, and the magnitude of change of primarily alanine aminotransferase activity at 1000 mg/kg, in combination with other clinical biochemistry changes that point to a disturbance in liver function, a No Observed Adverse Effect Level (NOAEL) for the test material of 300 mg/kg was established.