Methanesulfonic acid, 1,1'-(1,6-hexanediyldiimino)bis[1-oxo-, sodium salt (1:2)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic 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:

other: Study conducted according to OECD TG 408 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methansulfonic acid (1,6-hexanediyl-diimino)bis[1-oxo, disodium salt

EC Number:

690-526-2

Cas Number:

38632-47-2

Molecular formula:

C8H14S2N2O8.Na2

IUPAC Name:

Methansulfonic acid (1,6-hexanediyl-diimino)bis[1-oxo, disodium salt

Details on test material:

Purity/Concentration used for Formulation: 91.4 %. Purity was corrected after reanalysis (Certificate of Analysis LNX31603, 06 February 2012) to 84.7 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHanTM: WIST(SPF)
Age (at Delivery): Ca. 7 weeks
Body Weight Range (at Acclimatization): Males: 195 to 242 g (mean 219 g); Females: 123 to 149 g (mean 135 g)
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.
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 / dark cycle with ca. eight hours music during the light period.
Accommodation: In groups of two, three or four in Makrolon type-4 cages with wire mesh tops and standard softwood
bedding including paper enrichment.
Diet: Pelleted standard Harlan Teklad 2914C rat / mouse maintenance diet was available ad libitum. The feed batches were analyzed for contaminants. Results of respective analyses for contaminants are included in the report.
Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of
respective samples are included in the report.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Bidistilled

Details on oral exposure:

Frequency of Administration: Daily.
Dose Volume: 10 mL/kg body weight

Details on analytical verification of doses or concentrations:

The dose formulations were prepared to based upon the originally reported purity of 91.4%. However, a later Certificate of Analysis (LNX31603, from 06 Feb 2012) provided by the sponsor defined the purity as 84.7%.
The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a dose range finding study, the stability of the test item formulations was considered to be sufficient to justify.
After experimental start and during weeks 6 and 13, 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 test item was used as analytical standard. The results of analysis were summarized in an appendix and attached to the report.

Duration of treatment / exposure:

Duration of Pre-Randomization Phase: 1 day
Duration of Acclimatization Phase: 7 days
Duration of Treatment Phase: 91/92 days

Frequency of treatment:

Frequency of Administration: Daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day (control group)
Basis:
other: cotrol group

Doses / concentrations 2

Remarks:

Doses / Concentrations:
93 mg/kg/day
Basis:
other: actual ingested based on corrected purity of 84.7 %

Doses / concentrations 3

Remarks:

Doses / Concentrations:
278 mg/kg/day
Basis:
other: actual ingested based on corrected purity of 84.7 %

Doses / concentrations 4

Remarks:

Doses / Concentrations:
927 mg/kg/day
Basis:
other: actual ingested based on corrected purity of 84.7 %

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

According to OECD TG 408

Positive control:

No applicable

Examinations

Observations and examinations performed and frequency:

Daily Observations: The animals were observed for clinical signs once before commencement of administration
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 12) thereafter.
Food Consumption The food consumption was recorded once during the acclimatization period and weekly thereafter.
Body Weights: Body weights were recorded weekly during acclimatization and treatment periods and before necropsy.
Functional Observational Battery: During week 13, a modified Irwin screen test were evaluated in all animals.
Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge.
Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively. 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.
Clinical Laboratory Investigations after 13 Weeks: 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. Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.
Hematology: 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. Coagulation Parameter: Prothrombin time, Activated partial thromboplastin time.
Clinical Biochemistry: Glucose, Urea, Creatinine, Bilirubin total, Bile acids, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein total, Albumin, Globulin, Albumin/Globulin Ratio.
Urinalysis: Volume, Relative density, Color, Appearance, pH, Nitrite, Protein, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leucozytes.

Sacrifice and pathology:

All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded.
The following organs from all 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.: Adrenal glands, Brain, Epididymides, Heart including auricles, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus,
The following organs were collected and histopathologically examined: Adrenal glands, Brain - including section of medulla/pons,
cerebral and cerebellar cortex, Aorta, Bone marrow (sternum), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes w/optic nerve (fixed in Davidson's
solution), Heart including auricles, Ileum, with Peyer's patches
Jejunum, Kidneys, Laryn, Liver, Lungs, filled w/formalin at necropsy, Lymph nodes – mesenteric and mandibular, Mammary gland area, Ovaries, Pancreas, Pituitary gland, Prostate gland and seminal vesicles incl.coagulating glands, Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Skeletal muscle, Skin, Spinal cord - cervical, midthoracic, lumbar, Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Trachea, Urinary bladder, filled w/formalin at necropsy, Uterus (incl. oviducts, cerviand vagina), All gross lesions
The following organs were collected but not histopathologically investigated: Bone (sternum, femur including joint), Harderian gland (fixed in Davidson's solution), Lacrimal gland, exorbital, Nasal cavity, Pharyn, Tongue, Ureters.

Statistics:

Statistical Analysis: 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 (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 (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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the mean body weights were significantly reduced (p<0.05) in males from days 43 to 71 of treatment.

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were restricted to elevated bile acid levels in males and females at the highest dose level of 1000 mg/kg/day.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery, Grip Strength, Locomotor Activity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg: kidney, testes and brain weights were increased.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg: Spleen, Thymus

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Slightly lower body weights and kidney histopathology in males.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mortality / Viability

One male rat (no. 22 at 278 mg/kg/day) died on day 7 and one female rat (no. 76 at 927 mg/kg/day) died on day 47, and the macroscopical findings noted in these rats are generally associated with dosing error. All remaining animals survived the scheduled treatment period.

Clinical Signs (Daily and Weekly)

Test item-related clinical symptoms (slightly soft feces) were noted during daily observations in males and females at the highest dose level only. There were no clinical symptoms noted during the weekly behavioral observations (weeks 1-12).

Functional Observational Battery

There were no clinical symptoms noted during the functional observational battery performed during week 13 of treatment.

Grip Strength

The mean fore- and hind limb grip strength values of the test item-treated males and females were similar to those of the respective controls.

Locomotor Activity

There were no test item-related differences in the mean locomotor activity at any dose level.

Food Consumption

The mean daily food consumption values of the test item-treated male or female rats were similar to those of the respective control rats.

Body Weights

At 927 mg/kg/day, the mean body weights were significantly (approx. 10%) reduced (p<0.05) in males from days 43 to 71 of treatment. Although the differences noted from days 78 to 91 of treatment no longer attained statistical significance, they remained below those of the control males and were considered to be a test item-related effect. Females at this dose level did not have differences that attained statistical significance but remained below that of the control values (approx. 5%). The mean body weight gain values of the males was reduced from day 15 onwards, with statistically significant reductions appearing from days 43 to 71. The females’ body weight gain was marginally lower than that of the control females but without statistical significance.

At 278 mg/kg/day, the mean body weights of the males were generally similar to those of the controls males. Females showed similarly marginally, statistically not significant reduced values as were seen in the females treated with 927 mg/kg/day.

At 93 mg/kg/day, the body weights of males and females compared favorably with those of the controls.

Ophthalmoscopic Examinations

There were no test item-related differences.

Clinical Laboratory Investigations

Hematology

The hematology parameters showed no test item-related changes of toxicological relevance at any dose level.

Males treated with 978 mg/kg/day had significantly reduced methemoglobin levels, a finding generally not associated with toxicological relevance. In addition, the relative prothrombin time was significantly higher (p<0.05) than the control males. In the absence of a clear dose-response relationship or any prolongation in the activated partial thromboplastin time, this finding was

considered to be of no toxicological relevance.

Females at all dose levels showed significantly prolonged relative prothrombin times (either p<0.05 or p<0.01) when compared with the control females. As in the males, there was no clear dose-response relationship and the activated partial thromboplastin times were largely unchanged.

At 927 mg/kg/day, the mean platelet count was significantly reduced in females (p<0.01) when compared with the controls, but remained well within the range of the historical control values for this parameter.

Clinical Biochemistry

Test item-related changes were restricted to elevated bile acid levels in males and females at the highest dose level of 927 mg/kg/day. All other differences were within the ranges of the historical control values or were unrelated to dose.

Urinalysis

The urinalysis parameters showed no test item-related changes of toxicological relevance. All differences remained within the ranges of the historical control values.

Organ Weights

No test item-related changes of toxicological relevance were noted at any dose level because the observations were not dose dependent, in general slight changes are reported within the historical control values and not correlated to histopathologic findings. At 927 mg/kg/day, significantly elevated mean absolute brain weight (8%) and mean absolute brain-to-body weight ratio were noted when compared with the control males. The mean absolute kidney weight (15%)of these males was also significantly elevated as was the mean kidney-to-body weight ratio when compared with the controls. The mean absolute testes weight (14%) and mean testes-to-body weight ratio were significantly elevated. The mean absolute and relative organ weights of the test item-treated females were unaffected.

At 278 mg/kg/day, significantly elevated mean absolute brain weight (6%) was noted when compared with the control. No other differences were noted in the mean absolute or relative organ weight in males at this dose level. The mean absolute and relative organ weights of the females were unaffected.

At 93 mg/kg/day, significantly elevated mean absolute brain weight (6%) was noted when compared with the control. The mean absolute testes weight (11%) and mean testes-to-body weight ratio were significantly elevated. In females, the mean absolute liver weight was significantly elevated (10%). No other differences in organ weight or ratios were noted in this group.

Macroscopic / Microscopic Findings

Of the rats that survived until scheduled necropsy, there were no macroscopical findings that were test item-related. The macroscopical findings recorded were of varying incidence/severity and unrelated to dose.One male rat (no. 22 at 278 mg/kg/day) and one female rat (no. 76 at 927 mg/kg/day) died spontaneously on days 7 and 47, respectively. Macroscopical findings included reddish discoloration of incompletely collapsed lungs and were considered to indicate dosing errors. In addition to these changes, yellowish discoloration of the pyloric mucosa of the stomach and reddish discoloration of the mandibular lymph nodes were noted in the male rat.

Of the rats that survived until scheduled necropsy, there were no macroscopical findings that were clearly test item-related. The macroscopical findings which were recorded were of varying incidence/severity and unrelated to dose. Discoloration or foci of the stomach, pancreas, thymus, spleen or ovaries was noted in individual animals, as was enlargement of liver (in three males at

93 mg/kg/day), spleen and testes of males in various groups.

Microscopic Findings

Liver: Centrilobular to diffuse hepatocellular hypertrophy was recorded at minimal to slight severity in

males of dose group 2-4.

Finding / Groups 1 (10) M (10) F; 2 (10) M (10)3; F (10) M (10) F 4; (10) M (10) F

Hepatocellular hypertrophy - number of findings (severity)

Group 1: 0/10 M 0/10 F

Group 2: 4(1.3)/10 M; 0/10 F

Group 3: 7/(1.3)/10 M, 0/10 F

Group 4: 6(1.2)/10 M, 0/10 F

Since there is no dose response in the incidence or severity of the observation this observation is not regarded as compound related.

Kidneys: Increased mean severity of hyaline droplets along with increased incidence and mean severity of tubular basophilia was recorded in group 4 males.

Finding / Groups 1 (10) M (10) F; 2 (10) M (10)3; F (10) M (10) F 4; (10) M (10) F

Hyaline droplets

Group 1: 10 (1.0)/10 M, 0/10 F

Group 2: 10 (1.2)/10 M, 0/10 F

Group 3: 9 (1.4)/10 M, 0/10 F

Group 4: 8 (1.8)/10 M, 0/10 F

Tubular basophilia

Group 1: 9 (1.1)/10 M, 5(1.0)/10 F

Group 2: 10 (1.2)/10 M, 0/10 F

Group 3: 10 (1.2)/10 M, 0/10 F

Group 4: 10 (2.0)/10 M, 4 (1.0)/10 F

Hyaline droplets nephropathy was found in males treated with 927 mg/kg/day. The nephropathy consisted of elevated incidence mean severity of hyaline droplets along with tubular basophilia. However, the increased severity was statistically not significant. Therefore, the toxicological of the nephropathy is not clear from this study. Since the hyaline droplets in the male rat relate to accumulation of alpha-2-microglobulin and little or none of this protein is present in man, a nephropathy in man that involves the same mechanism is unlikely to occur with the test item. Although this finding is adverse during exposure for the male rat it has little or no relevance for other species including man.