3-methoxy-N,N-dimethylpropionamide

Endpoint summary

Description of key information

A 28-day oral repeated dose toxicity study was conducted with test substance according to OECD 407 guideline. The NOAEL was determined to be 25 mg/kg bw/day based on increased liver weights, enlargement of the liver and higher level of total cholesterol in both sexes at 150 and 1000 mg/kg bw/day.

A 90-day oral repeated dose toxicity study was conducted with test substance according to OECD 408 guideline. The NOAEL of test substance was determined to be 10 mg/kg bw/day based on the increases in the liver and kidney weights accompanied by histopathological and serum chemistry changes at 100 and/or 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 September 1998

Conclusions:

In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD 408 guideline and in compliance with GLP principles, the NOAEL was found to be 10 mg/kg bw/day, based on increased liver and kidneys weights and associated histopathological and serum chemistry changes at 100 and/or 1000 mg/kg bw/day.

Executive summary:

A 90-day oral repeated dose toxicity study was conducted with test substance according to OECD 408 guideline and in compliance with GLP principles. Male and female rats were exposed to 0, 10, 100 or 1000 mg/kg bw/day via oral gavage. Chemical analysis of the formulations confirmed correct concentrations, homogeneity and stability. No mortality occurred during the study. No clinical signs or toxicologically significant changes in functional observations, opthalmoscopy, haematology parameters, urinalysis and oestrus cycle phases were noted.

During the study period minor reductions in body weight gain were noted at 1000 mg/kg bw/day towards the end of the treatment period after days 63 (males)/70 (females) which were not associated with changes in food consumption. These variations did not reach statistical significance, except for the body weight loss in females at week 13 (-2.0 g, p<0.05) and did not show a clear dose response, however a possible treatment related effect on body weight gain at 1000 mg/kg bw/day towards the end of the treatment period cannot be excluded.

No test item related changes were noted at the macroscopic examination on day 90. Increases in liver weights were noted. Relative liver weights (adjusted for bw) were increased up to 56% in males and up to 60% in females at the highest dose level of 1000 mg/kg bw/day, attaining statistical significance at all dose levels in males and at 1000 mg/kg bw/day in females. Histopathological findings in the liver included centrilobular heptacellular hypertrophy in the 100 mg/kg bw/day animals (minimal degree 6/10 males, 2/10 females) and in the 1000 mg/kg bw/day animals (mild degree: 6/10 males, 5/10 females; minimal degree: 4/10 males, 5/10 females). Centrilobular hepatocellular vacuolation was also noted in males at 100 mg/kg bw/day (minimal degree 2/10) and at 1000 mg/kg bw/day (4/10 minimal, 3/10 mild and 1/10 moderate). In absence of histopathological findings, the liver weight changes noted at 10 mg/kg bw/day in males were not considered adverse; furthermore, although statistically significant, the relative liver weight increase was below 10% (6%) at this dose level. The histopathological finding in the liver at 100 and 1000 mg/kg bw/day were considered to be associated with administration of the test item.

For relative kidneys weight increases up to 26% and 22% were noted in males and females, respectively, attaining statistical significance at the 1000 mg/kg bw/day dose level only. Histopathological findings in the kidney included tubular basophilia (with predominantly minimal severity) in 10/10 males and 2/10 females at the 1000 mg/kg bw/day dose levels and 4/10 males at the 100 mg/kg bw/day dose level observed together with mononuclear infiltrate in high dose animals (1000 mg/kg bw/day). Minimal focal cortical atrophy with mononuclear cell infiltrate was also present in one high dose male. Mild to moderate intracellular eosinophilic droplets were present in animals at the 1000 mg/kg bw/day dose levels (10/10 males and minimal in 1/10 females) and minimal to mild tubular proteinaceous casts were noted at the 100 mg/kg bw/day (1/10 males, 4/10 females) and 1000 mg/kg bw/day (5/10 males, 4/10 females) dose levels. Additionally, minimal tubular basophilia of the right kidney without cell infiltrate was noted in 3/10 control males. Based on the histopathological findings the observed changes at 100 and 1000 mg/kg bw/day were considered to be associated with administration of the test item.

No toxicologically significant changes on the clinical chemistry parameters were noted at the 10 mg/kg bw/day dose level. Statistically significant increases in mean cholesterol values compared to controls were noted in 100 and 1000 mg/kg bw/day animals (51.6% and 100.2% higher than in controls in males and 26.4% and 109% higher than in controls in females, respectively). Total bilirubin concentrations were also increased in the 1000 mg/kg bw/day animals (33.9% higher than in controls in males (p<0.01) and 22.4% higher than in controls in females (p<0.05)). These changes at the 100 and 1000 mg/kg bw/day dose levels were considered related to test item administration. In addition, minor variations in the absence of a clear dose response were noted in the sodium (Na+ up to 1.7% lower than in controls (100 mg/kg bw/day)), chloride (Cl- up to 3% lower than in controls (100 mg/kg bw/day males)), calcium (Ca2+ up to 9.2% higher than in controls (1000 mg/kg bw/day females)) and phosphorus mean serum concentration (Phos up to 46.8% higher than in controls (1000 mg/kg bw/day females)) when compared to control values, which in view of the histological findings in the liver and/or kidneys, cannot be excluded to be test item related.

Taken all data together, the NOAEL of test substance was determined to be 10 mg/kg bw/day based on the increases in the liver and kidney weights accompanied by histopathological and serum chemistry changes at 100 and/or 1000 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2007 - 12 November 2007

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:

27 July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 September 1996

Deviations:

no

Principles of method if other than guideline:

"28-day repeated oral dose toxicity study using mammals" specified in the "Methods of test relating to a new chemical substance" (Yakushoku-hatsu No.1121002, Heisei 15.11.13 Seikyoku No.2, Kanpoki-hatsu No.031121002, November 21, 2003),

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamou-gun, Shiga 529-1633, Japan)
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 5 weeks
- Weight at study initiation: males: 146.4 - 170.1g; females: 112.1 - 142.2g
- Fasting period before study: No information available.
- Housing: The animals were housed in groups of 3 or 5 in stainless steel wire-mesh floor cages before group assignment and individually in stainless steel wire-mesh floor cages after group assignment.
- Diet: Free access to pellet diet (MF, Lot No. 070607, Oriental Yeast Co., Ltd.)
- Water: Free access to tap water supplied by Hita city (added chlorine)
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
With regard to the pellet diet, the results of the analysis conducted by the Japan Food Research Laboratories were acquired from the manufacturer and compared with the standard of Hita Laboratory established by referring to "Limits for Contaminants in Feed and Vehicles" in the United States Environmental Protection Agency Toxic Substances Control Act (1979), and only those lots meeting the standard were used. Drinking water was analyzed for contaminants by the Oita Pharmacist Association twice a year (but the taste test was conducted by Hita Laboratmy) to assure that the data for all of the parameters are within the standard values of Hita Laboratory established by referring to the water quality standard described in the "Ministerial Ordinance Concerning Water Quality Standard" (Ministry of Health, Labor and Welfare ordinance No. 101). It had been assured that all data were within the standard values.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 - 24.0
- Humidity (%): 48.8 - 64.1
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 August 2007 To: 27 September 2007

Route of administration:

oral: gavage

Details on route of administration:

The dosing solution of the test substance was administered daily in the morning for 28 days by gavage administration using a syringe (Tenuno Corporation) connected to a Nelaton catheter (Terumo Corporation). Thereafter, the recovery period was established for 14 days.

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighed, and olive oil was added to prepare the 10.0 w/v% formulation. The 0.25 and 1.5 w/v% formulations were prepared by diluting this 10.0 w/v% formulation with olive oil in a serial dilution method. The preparation was conducted once for 7 or 8 days under a sodium-light. The formulations were stored in a cool and dark place.

VEHICLE
From the information that the test substance was hydrolysable, 10.0 w/v% formulation of the test substance in olive oil was investigated. Since the test substance was soluble, olive oil (Lot number: 041OMA, Fujimi Pharmaceutical) was used as the vehicle for this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of the concentration of the test substance in dose formulation was performed at the start of administration period in the target study. Samples were collected from the middle layers of the dose formulations (n=1), and each sample was pretreated and analyzed once by GC. Test substance and dose formulation were treated under light protection and subjected to the pretreatment.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Daily in the morning for 28 days.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Recovery group

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 7-day repeated oral administration toxicity study was conducted with 4 dose levels of 25, 250, 500 and 1000 mg/kg bw/day. As result, increased liver weight and enlargement liver were observed in the group of 500 mg/kg bw/day or more. Therefore, for the main study, the three dose levels of 25, 150 and 1000 mg/kg bw/day as the high dose level were selected.
- Recovery groups were selected for the 1000 mg/kg bw/day and vehicle control groups.

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before the starting day of administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 3 times a day (before administration, during-immediately after administration and in the afternoon) during the administration period. During the recovery period, observation was made in the morning and in the afternoon everyday.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at the time of group assigmnent during pre-treatment period and on days 1, 3, 8, 12, 17, 21, 26 and 28 during the administration period, as well as on days 1, 5, 10 and 14 during the recovery period. Animals were also weighed once at removal for necropsy from the cage after the end of the administration period and once after the end of the recovery period for the calculation of relative organ weight.

FOOD EFFICIENCY:
- Food consumption was measured at the time of group assignment during pre-treatment period and on days 1, 3, 8, 15, 22 and 28 during the administration period, as well as on days 1, 4, 8 and 14 during the recovery period. The mean daily consumption was calculated based on the cumulative value from a measurement day to the next measurement day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After the administration period and after the recovery period.
- Anaesthetic used for blood collection: Yes, ether anesthesia.
- Animals fasted: Yes, overnight (16-20 hours)
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: After the administration period and after the recovery period.
- Animals fasted: Yes, overnight (16-20 hours)
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines.

URINALYSIS: Yes
- Time schedule for collection of urine: On the last day of the administration period (except for the animals in the recovety groups) and of the recovery period, and 15 to 17-hour accumulated urine samples were collected under free access to drinking water.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: According to test guidelines.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At week 4 of the administration period. Since no abnormalities were detected at week 4 of the administration period, the functional examination at week 2 of the recovery period was not performed.
- Dose groups that were examined: all rats/sex/group
- Battery of functions tested: reflex test / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: The kidneys were examined after special staining.
- How many animals: 4 males
- Dose groups that were examined: Vehicle and test substance groups
- Parameters checked: Immunohistochemistry with anti-α2u-globulin antibody.

Sacrifice and pathology:

GROSS PATHOLOGY: All animals were subjected to macroscopic examination including the observation of the body surface, orifice, cranial cavity, thoracic cavity, abdominal cavity and contents thereof.

ORGAN WEIGHTS:
The weights of the organs listed below were measured in all animals. In addition, the relative weight per 100 g body weight was calculated for all organs based on the body weight at the time of removal for necropsy from the cage.
Liver (g), heart (g), kidney* (g), testis* (g), epididymis*(g), ovary* (mg), brain (g), spleen (g), thymus (mg), adrenal gland* (mg)
* Right and left organ weights combined

HISTOPATHOLOGY: The organs and tissues listed below were collected from all animals. The collected organs and tissues were fixed in 10% neutral buffered formalin solution. The testis and epididymis were fixed in Bouin's solution.
Organ/tissue: Trachea and lung, stomach, intestine (including duodenum-rectum and Peyer's patch), liver, heart, kidney, urinary bladder, testis, epididymis, prostate gland, seminal vesicle, ovary, uterus, vagina, brain (including cerebrum, cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), lymph node (axillary and mesenteric lymph nodes), spleen, thymus, pituitary gland, thyroid gland (including parathyroid), adrenal gland, eye ball.
As the macroscopic lesions, skin was additionally collected.
From the organs and tissues, paraffin-embedded thin sections were prepared, stained with hematoxylin and eosin (HE), and then examined microscopically.

Statistics:

The results concerning body weight (except for the weight at removal for necropsy from the cage), food consumption, hematological examination, blood chemical examination, urine volume, urine specific gravity, organ weight, grip strength and spontaneous motor activity were tested for homogeneity of variance using the Bartlett test, and when the variance was homogeneous at 5 % significance level, one-way analysis of variance was performed. When there was a significant difference in the test for homogeneity of variance, between-group test for the vehicle control group and test substance group was performed using the Dunnett's test.
When the variance was not homogeneous, the Kruskal-Wallis test was performed, and when there was a significant difference in the test, between-group test for the vehicle control group and test substance group was performed using the nonparametric Dunnett's test.
The results concerning the number of defecation (feces count) and the number of urination (number of urine pool) were tested using the Kruskal-Wallis test, and when there was a significant difference in the test, between-group test for the vehicle control group and test substance group was performed using the nonparametric Dunnett's test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs:
Administration period:
Male: Salivation after administration was observed in 2, 3, 4 and 10 animals of the vehicle control, 25, 150 and 1000 mg/kg bw/day groups, respectively. In addition, 4 animals of the 1000 mg/kg bw/day group showed decreased motor activity after administration from day 20 to 27.
Female: Salivation after administration was observed in 2, 1, 2 and 8 animals of the vehicle control, 25, 150 and 1000 mg/kg bw/day groups, respectively. In addition, 1 animal of the 1000 mg/kg bw/day group showed hair loss and eschar formation.
Recovery period:
Male: No abnormalities were detected.
Female: One animal of the 1000 mg/kg bw/day group showed hair loss and eschar formation which was continued from the administration period.
Detailed clinical observation:
Administration period and recovery period: No abnormalities were detected for any observation item in male and female animals.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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):

At the end of administration period:
Male: Prothrombin time and activated partial thromboplastin time were significantly prolonged in the 1000 mg/kg bw/day group.
Female: Significantly higher values of platelet count and white blood cell count were observed in the group of 150 mg/kg bw/day or more and in the 1000 mg/kg bw/day group, respectively.
At the end of recovery period:
Male: No abnormalities were detected.
Female: Significantly higher values of red blood cell count and hematocrit, and a significantly lower value of mean corpuscular hemoglobin concentration were observed in the 1000 mg/kg bw/day group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period:
Male: Significantly lower levels of albumin and chlorine in the 150 mg/kg bw/day group and a significantly higher level of total cholesterol in the group of 150 mg/kg bw/day or more were observed. Significantly higher levels of alanine aminotransferase, total protein and calcium, and a significantly lower level of A/G ratio were observed in the 1000 mg/kg bw/day group.
Female: Significantly lower and higher levels of alkaline phosphatase and total cholesterol were observed in the group of 150 mg/kg bw/day or more, respectively. Significantly higher levels of alanine aminotransferase and significantly lower levels of A/G ratio were observed in the 1000 mg/kg bw/day group, respectively.
At the end of recovery period:
Male: No abnormalities were detected.
Female: A significantly lower level of creatinine was observed in the 1000 mg/kg bw/day group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of administration period:
Male: Three and 4 animals showed increased epithelial cell and urinary cast in the 150 mg/kg bw/day group, respectively. Four animals showed increased epithelial cell and urinary cast in the 1000 mg/kg bw/day.
Female: No abnormalities were detected.
At the end of recovery period:
No abnormalities were detected for any parameter in male and female animals.

Behaviour (functional findings):

no effects observed

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of administration period:
Male: In the 1000 mg/kg bw/day group, increased eosinophilic bodies and hyaline droplets in the kidney showed positive reaction to anti-α2u-globulin antibody.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period:
Male: Significantly higher value of absolute kidney weight was observed in the 25 and 150 mg/kg bw/day group. Significantly higher value of relative weight for liver and kidney in the group of 150 mglkg or more and significantly higher value of absolute weight for liver in the group of 1000 mg/kg bw/day were observed.
Female: Significantly higher values of relative weight of the liver was observed in the group of 150 mg/kg bw/day or more. Significantly higher value of absolute weight for liver and ovary and absolute and relative weight for kidney in the group of 1000 mg/kg bw/day were observed.
At the end of recovery period:
Male: No abnormalities were detected for any organ weight.
Female: Significantly higher value of relative thymus weight was observed in the 1000 mg/kg bw/day group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period:
Male: In the 25 mg/kg bw/day group, apparent spotty pattern of the kidney surface was observed in 2 animals and small-sized soft testis with small-sized epididymis in one animal. In the 150 mg/kg bw/day group, enlargement of the liver, apparent spotty pattern of the kidney surface, small-sized testis and epididymis, and decreased size of left lobe in the thyroid gland were observed in 3, 4, one and one animals, respectively. In the 1000 mg/kg bw/day group, enlargement of the liver and apparent spotty pattern of the kidney surface were observed in 5 animals and enlargement of the kidney with recessed region was observed in one animal.
Female: Whitish region on spleen capsule and blackish region of mucosal membrane in the glandular stomach were observed in one animal in the 25 mg/kg group and one animal in the 150 mg/kg bw/day group, respectively. Enlargement of the liver were observed in 2 animals in the 150 mg/kg bw/day group and 5 animals in the 1000 mg/kg bw/day group.
At the end of recovery period:
Male: Diverticulum of the jejunum was observed in one animal in the vehicle control group.
Female: One female in the 1000 mg/kg bw/day group showed skin erosion and sparse fur.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period:
Male: In the vehicle control group, focal necrosis of hepatocytes in the liver was observed in one animal. In the 25 mg/kg bw/day group, increased eosinophilic bodies in the kidney was observed in 2 animals, diffuse atrophy of seminiferous tubles in the testis, decreased spermatozoa and germ cell debris in the lumen of the epididymis were observed in one animal. In the 150 mg/kg bw/day group, hypertrophy of centrilobular hepatocytes in the liver, microgranuloma in the liver, increased eosinophilic bodies in the kidney, degeneration of spermatocytes in the testis, decreased spermatozoa and germ cell debris in the lumen of the epididymis and hypoplasia of left lobe in the thyroid gland were observed in 4, 2, 3, 4, one and one animals, respectively. In the 1000 mg/kg bw/day group, hypertrophy of centrilobular hepatocytes in the liver, basophilic tubules in the kidney, increased eosinophilic bodies in the kidney and increased hyaline droplets in the kidney were observed in 5, one, 4 and 5 animals, respectively. Increased eosinophilic bodies and hyaline droplets in the kidney showed positive reaction to anti-α2u-globulin antibody.
Female: In the 25 mg/kg bw/day group, capsulitis in the spleen was observed in one animal. In the 150 mg/kg bw/day group, necrosis of fundic mucosa in the glandular stomach and microgranuloma in the liver were observed in one and one animal, respectively. In the 1000 mg/kg bw/day group, hypertrophy of centrilobular hepatocytes in the liver and mineralization in cortico-medullary junction in the kidney were observed in 5 and one animals, respectively.
At the end of recovery period:
Male: In the vehicle control group, diverticulum of the jejunum and microgranuloma in the liver were observed in one and one animals, respectively. In the 1000 mg/kg bw/day group, hypertrophy of centrilobular hepatocytes in the liver and microgranuloma in the liver were observed in 2 and one animals, respectively.
Female: In the 1000 mg/kg bw/day group, microgranuloma in the liver and skin ulcer were observed in 2 and one animals, respectively.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Confirmatory study on the stability of MDMPA and homogeneity, stability and concentration of dose formulation:

The test substance (MDMPA) was stable throughout the administration period in the target study.

The test substance in 10.0 and 0.01 w/v% dose formulations possessed an excellent homogeneity and was stable for 8 days after preparation when stored in a cool and dark place. It was also confirmed

that the 10.0, 1.5 and 0.25 w/v% dose formulations for the target study were prepared at concentrations nearly identical to the nominal concentrations.

The value of the relative ratio was 99.2~100% for 10.0, 1.5 and 0.25 w/v% dose formulations and met the judgment criterion.

Conclusions:

In a 28-day oral repeated dose toxicity study with rats, conducted according to OECD 407 guideline and in compliance with GLP principles, the NOAEL was found to be 25 mg/kg bw/day, based on increased liver weights, enlargement of the liver and higher level of total cholesterol in both sexes at 150 and 1000 mg/kg bw/day.

Executive summary:

A 28-day oral repeated dose toxicity study was conducted with test substance according to OECD 407 guideline and in compliance with GLP principles. Male and female rats (5/sex/dose) were exposed via oral gavage to 0, 25, 150 or 1000 mg/kg bw/day, and to 0 and 1000 mg/kg bw/day for the recovery. Chemical analysis of the formulations confirmed correct concentrations, homogeneity and stability. No mortality occurred during the study. No clinical signs or toxicologically significant changes in body weight, food efficiency, functional observations, urinalysis, immunological findings and neuropathological findings were noted.

Enlargement of the liver was observed in both sexes at 150 and 1000 mg/kg bw/day at necrospsy. Increased relative liver weight in both sexes at 150 and 1000 mg/kg bw/day and increased absolute liver weight in both sexes at 1000 mg/kg bw/day was observed. In the histological examination, hypertrophy of centrilobular hepatocytes in the liver was observed in the males at 150 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.

The hematological examination revealed elongations of prothrombin time and activated partial thromboplastin time in males at 1000 mg/kg bw/day.

In the blood chemical examination, higher level of total cholesterol was observed in both sexes at 150 and 1000 mg/kg bw/day. In addition, in both sexes at 1000 mg/kg bw/day exhibited high and low levels of alanine aminotransferase and A/G ratio, respectively. Males at 1000 mg/kg bw/day also showed a high level of total protein. These indicated the effects on the liver. Though the high level of total protein was observed only in males, the effect of the substance cannot be excluded because the A/G ratio was affected.

In the recovery group, males of the 1000 mg/kg bw/day group showed hypertrophy of centrilobular hepatocytes in the liver. However, the severity and the incidence were less than those at the end of the administration period. In addition, the changes in the liver of females were not detected and no abnormality of blood coagulation time was observed.

Based on the results, the NOAEL of test substance was determined to be 25 mg/kg bw/day based on increased liver weights, enlargement of the liver and higher level of total cholesterol in both sexes at 150 and 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The repeated oral toxicity results are of sufficient quality and adequate for this dossier.

Organ:

kidney

liver

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

Repeated dose 28 Day:

A 28-day oral repeated dose toxicity study was conducted with test substance according to OECD 407 guideline and in compliance with GLP principles. Male and female rats (5/sex/dose) were exposed via oral gavage to 0, 25, 150 or 1000 mg/kg bw/day, and to 0 and 1000 mg/kg bw/day for the recovery. Chemical analysis of the formulations confirmed correct concentrations, homogeneity and stability. No mortality occurred during the study. No clinical signs or toxicologically significant changes in body weight, food efficiency, functional observations, urinalysis, immunological findings and neuropathological findings were noted.

Enlargement of the liver was observed in both sexes at 150 and 1000 mg/kg bw/day at necrospsy. Increased relative liver weight in both sexes at 150 and 1000 mg/kg bw/day and increased absolute liver weight in both sexes at 1000 mg/kg bw/day was observed. In the histological examination, hypertrophy of centrilobular hepatocytes in the liver was observed in the males at 150 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.

The hematological examination revealed elongations of prothrombin time and activated partial thromboplastin time in males at 1000 mg/kg bw/day.

In the blood chemical examination, higher level of total cholesterol was observed in both sexes at 150 and 1000 mg/kg bw/day. In addition, in both sexes at 1000 mg/kg bw/day exhibited high and low levels of alanine aminotransferase and A/G ratio, respectively. Males at 1000 mg/kg bw/day also showed a high level of total protein. These indicated the effects on the liver. Though the high level of total protein was observed only in males, the effect of the substance cannot be excluded because the A/G ratio was affected.

In the recovery group, males of the 1000 mg/kg bw/day group showed hypertrophy of centrilobular hepatocytes in the liver. However, the severity and the incidence were less than those at the end of the administration period. In addition, the changes in the liver of females were not detected and no abnormality of blood coagulation time was observed.

Based on the results, the NOAEL of test substance was determined to be 25 mg/kg bw/day based on increased liver weights, enlargement of the liver and higher level of total cholesterol in both sexes at 150 and 1000 mg/kg bw/day.

Repeated dose 90 Day:

A 90-day oral repeated dose toxicity study was conducted with test substance according to OECD 408 guideline and in compliance with GLP principles. Male and female rats were exposed to 0, 10, 100 or 1000 mg/kg bw/day via oral gavage. Chemical analysis of the formulations confirmed correct concentrations, homogeneity and stability. No mortality occurred during the study. No clinical signs or toxicologically significant changes in functional observations, opthalmoscopy, haematology parameters, urinalysis and oestrus cycle phases were noted.

During the study period minor reductions in body weight gain were noted at 1000 mg/kg bw/day towards the end of the treatment period after days 63 (males)/70 (females) which were not associated with changes in food consumption. These variations did not reach statistical significance, except for the body weight loss in females at week 13 (-2.0 g, p<0.05) and did not show a clear dose response, however a possible treatment related effect on body weight gain at 1000 mg/kg bw/day towards the end of the treatment period cannot be excluded.

No test item related changes were noted at the macroscopic examination on day 90. Increases in liver weights were noted. Relative liver weights (adjusted for bw) were increased up to 56% in males and up to 60% in females at the highest dose level of 1000 mg/kg bw/day, attaining statistical significance at all dose levels in males and at 1000 mg/kg bw/day in females. Histopathological findings in the liver included centrilobular heptacellular hypertrophy in the 100 mg/kg bw/day animals (minimal degree 6/10 males, 2/10 females) and in the 1000 mg/kg bw/day animals (mild degree: 6/10 males, 5/10 females; minimal degree: 4/10 males, 5/10 females). Centrilobular hepatocellular vacuolation was also noted in males at 100 mg/kg bw/day (minimal degree 2/10) and at 1000 mg/kg bw/day (4/10 minimal, 3/10 mild and 1/10 moderate). In absence of histopathological findings, the liver weight changes noted at 10 mg/kg bw/day in males were not considered adverse; furthermore, although statistically significant, the relative liver weight increase was below 10% (6%) at this dose level. The histopathological finding in the liver at 100 and 1000 mg/kg bw/day were considered to be associated with administration of the test item.

For relative kidneys weight increases up to 26% and 22% were noted in males and females, respectively, attaining statistical significance at the 1000 mg/kg bw/day dose level only. Histopathological findings in the kidney included tubular basophilia (with predominantly minimal severity) in 10/10 males and 2/10 females at the 1000 mg/kg bw/day dose levels and 4/10 males at the 100 mg/kg bw/day dose level observed together with mononuclear infiltrate in high dose animals (1000 mg/kg bw/day). Minimal focal cortical atrophy with mononuclear cell infiltrate was also present in one high dose male. Mild to moderate intracellular eosinophilic droplets were present in animals at the 1000 mg/kg bw/day dose levels (10/10 males and minimal in 1/10 females) and minimal to mild tubular proteinaceous casts were noted at the 100 mg/kg bw/day (1/10 males, 4/10 females) and 1000 mg/kg bw/day (5/10 males, 4/10 females) dose levels. Additionally, minimal tubular basophilia of the right kidney without cell infiltrate was noted in 3/10 control males. Based on the histopathological findings the observed changes at 100 and 1000 mg/kg bw/day were considered to be associated with administration of the test item.

No toxicologically significant changes on the clinical chemistry parameters were noted at the 10 mg/kg bw/day dose level. Statistically significant increases in mean cholesterol values compared to controls were noted in 100 and 1000 mg/kg bw/day animals (51.6% and 100.2% higher than in controls in males and 26.4% and 109% higher than in controls in females, respectively). Total bilirubin concentrations were also increased in the 1000 mg/kg bw/day animals (33.9% higher than in controls in males (p<0.01) and 22.4% higher than in controls in females (p<0.05)). These changes at the 100 and 1000 mg/kg bw/day dose levels were considered related to test item administration. In addition, minor variations in the absence of a clear dose response were noted in the sodium (Na+ up to 1.7% lower than in controls (100 mg/kg bw/day)), chloride (Cl- up to 3% lower than in controls (100 mg/kg bw/day males)), calcium (Ca2+ up to 9.2% higher than in controls (1000 mg/kg bw/day females)) and phosphorus mean serum concentration (Phos up to 46.8% higher than in controls (1000 mg/kg bw/day females)) when compared to control values, which in view of the histological findings in the liver and/or kidneys, cannot be excluded to be test item related.

Taken all data together, the NOAEL of test substance was determined to be 10 mg/kg bw/day based on the increases in the liver and kidney weights accompanied by histopathological and serum chemistry changes at 100 and/or 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the histopathological and clinical chemistry observations that are made in the available 90d study, the kidney and the liver are identified as possible target organs. In order to determine the need for classification as a STOT RE toxicant, the following findings are taken into account:

Kidney:

The histopathological findings in the kidneys include tubular basophilia, tubular proteinaceous casts and eosinophilic droplets. The function of the renal tubules is to allow for the reabsorption of electrolytes from the tubular fluid back into the blood circulation. Toxicity to the renal tubules can thus lead to deviations in the blood concentration of specific electrolytes.

When looking at the clinical chemistry results in the available 90d study, indeed some degrees of hypochloremia, hyponatremia and hyperphosphatemia are observed. The clinical chemistry observations thus are in line with the histopathological findings.

It has however to be noted that for the purpose of STOT-RE classification, the severity of the observed effects should be indicative of functional impairment of the organ. Basophilia in renal tubules is a common observation in case of tubule regeneration. In the absence of additional histopathological findings, the observed effects are equivocal for renal dysfunction, and therefore insufficient to warrant STOT-RE classification.

Liver:

The gross and histopathological findings in the liver include increased relative liver weight, hepatocellular centrilobular hypertrophy and hepatocellular centrilobular vacuolation. The interpretation of observations of hepatocellular hypertrophy is discussed by the Food Safety Commission, who has published its final decision on the matter in October 2017. The report describes that hypertrophy in itself can either be indicative of an adaptive response of the organism, or an indication of a true adverse effect of the chemical, depending on whether or not the homeostasis in the liver is maintained.

From the clinical biochemistry data in the available 90d study it is apparent that significantly increased cholesterol levels occur in the same dose groups that are also showing hepatocellular hypertrophy, thus indicating that the normal homeostasis in the liver is disturbed. As a consequence, the observed effects are to be considered as adverse effects leading to functional impairment of the organ. The effects occur in a dose-response related way at doses of 100 and 1000 mg/kg bw/d. According to CLP sections 3.9.2.7.3 c and f, the substance thus should be classified as STOT-RE 2, with the liver identified as target organ.

Reference: “Interpretation of the liver hypertrophy in the toxicological evaluation of veterinary medicinal products and feed additives.” The Food Safety Commission. Final decision on October 25, 2017.