2-ethyl-2-[(3-mercapto-1-oxopropoxy)methyl]propane-1,3-diyl bis[3-mercaptopropionate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Version / remarks:

30 September 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 September 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol tetrakis(3-mercaptopropionate)

EC Number:

231-472-8

EC Name:

Pentaerythritol tetrakis(3-mercaptopropionate)

Cas Number:

7575-23-7

Molecular formula:

C17H28O8S4

IUPAC Name:

3-[(3-sulfanylpropanoyl)oxy]-2,2-bis{[(3-sulfanylpropanoyl)oxy]methyl}propyl 3-sulfanylpropanoate

Details on test material:

- Name of test material (as cited in study report): PETMP
- Physical state: liquid
- Analytical purity: 97.4%, dose levels corrected for purity

Test animals

Species:

rat

Strain:

other: RccHan: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Ca. 7 weeks at delivery
- Weight at study initiation: Males: 162 - 203 g (mean: 181 g) / Females: 125 - 151 g (mean: 138 g)
- Housing: In groups of two to five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%):30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). The mixture was mixed thoroughly using a magnetic stirrer until homogeneous. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The dose formulations were stored for up to eight days in glass beakers in a refrigerator at 2 - 8 °C.


VEHICLE
- Concentration in vehicle: 0/ 2.5 / 10 / 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-sampling: at start, on days 1, 42 and 85 of treatment, duplicate samples of the control group as well as three samples (top, middle and bottom) were taken prior to dosing for analysis of homogeneity and concentration / duplicate samples were taken at experimental start to confirm stability (4 hour and 8 days)
- samples were stored there at -15 - 25 °C until analysis.
- analysis by HPLC coupled to a UV detector (215 nm)

Duration of treatment / exposure:

treatment: 91/92 days ; recovery: 28 d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 12.5, 50, 200 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 (control group), 10 (treatment groups), 5 (recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose range finding study
- Post-exposure recovery period in satellite groups: 4 weeks

- Dose selection rationale: based on results obtained in a 14-day dose range-finding study
- Rationale for animal assignment (if not random): Randomly allocated to groups by body weight.
- Post-exposure recovery period in satellite groups: 28 d

Examinations

Observations and examinations performed and frequency:

Viability / Mortality: Twice daily
Clinical signs: once daily during accimatisation; Twice daily on days 1 to 3; once daily thereafter; once daily during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly (weeks 1-12)

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (weekly)

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once in week 13 and 17 (recovery groups)
- Dose groups that were examined:
main + recovery groups: animals of the control and high dose groups as well as in animals of the middle dose groups if test item-related changes are seen in the high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: main + recovery groups: week 13 / recovery groups: week 17
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters: 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), total Leukocyte count, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes) Large unstained cells, Platelet count, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: main + recovery groups: week 13 / recovery groups: week 17
- Animals fasted: Yes
- How many animals: all
- Parameters: Glucose, Urea, Creatinine, toral Bilirubin, total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, total Protein, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: main + recovery groups: week 13 / recovery groups: week 17
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones , Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, Functional Observational Battery (Screen)
- Time schedule for examinations: last week of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: Grip Strength, Locomotor Activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopy, 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:

effects observed, treatment-related

Description (incidence and severity):

Daily Observations
Test item-related clinical signs were noted in both genders at 200 mg/kg bw/d on the first day of treatment.
Tachypnea was noted in two males and one female, whereas convulsions and/or shivering were noted in three males and two females. Prostration, hunched posture, ruffled fur and/or stiff gait were noted in three females. Pica was also observed in four males and one female at the highest dose level. With the exception of a few transient incidents, these signs were no longer seen after day 1. Because the findings were of limited incidence and brief duration, they were considered to indicate acute toxicity from a metabolic overload which triggered a subsequent adaptive response.
Salivation, noted from the third treatment week until the end of treatment, is a typical finding of discomfort noted in rats treated with a test item formulation of unpleasant taste.
At 50 mg/kg bw/day, the acute toxic response was not evident. Salivation began during treatment week 3 and persisted until the end of treatment.
At 12.5 mg/kg bw/day, intermittent findings included pica, ruffled fur, salivation, and reddish nasal secretion (typically porphyrin).

Incidental findings included weakened condition, areas of hair loss, localized scabbing, kinked tail and/or chromodacryorrhea) were noted infrequently (including one control female) and were therefore considered to be of no relevance. One control female had a palpable mass on the left shoulder in week 13 of treatment.
No late effects of toxicological relevance were noted during the recovery period.

Weekly Detailed Observations
No findings of toxicological relevance were noted in weeks 1 to 12 in male or female rats in test item-treated groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two premature decedents:
One male of the 12.5 mg/kg bw/d dose group was found dead on day 80 of treatment. In the 2 - 3 days prior to its death, clinical signs included weakened condition, ruffled fur and reddish nasal secretion.
One female of the 200 mg/kg bw/d dose group was sacrificed for ethical reasons on day 51 of treatment. This female could not move its hind limbs. Because it was unable to reach the food trough or water bottle, it was removed from the study.
All other animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean absolute and relative body weights of the test item treated rats were similar to those of the controls. No late effects were noted during the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated rats was not affected. No late effects were noted during the recovery period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related ophthalmoscopic changes. Typical background findings were noted (uni- or bilateral corneal opacities, persistent hyaloid vessel, persistent pupillary membrane, vitreous floaters) in test item-treated and control rats. The severity and incidence of these findings at the end of the treatment and recovery periods were similar.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the hematology parameters of males or females at any dose level. A small number of statistically significant but toxicologically irrelevant changes were noted at all dose levels. All differences were either within the ranges of the historical control data, not related to dose, or not accompanied by concomitant changes in related parameters and therefore considered to be incidental.
There were no late effects during the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Differences noted in mean levels of sodium, potassium and chloride of male rats treated with 50 mg/kg bw/day and 200 mg/kg bw/day were considered to be test item-related. These differences were statistically significant (p<0.05 or p<0.01); the potassium values exceeded the upper range of the historical control data. Females treated with 200 mg/kg/day had significantly higher mean potassium levels (p<0.01) and chloride levels (p<0.05). The potassium levels exceeded the upper range of the historical control data.
The statistically significant phosphorus values noted in females treated with 200 mg/kg bw/day (p<0.01) were within the range of the historical control data and considered to be incidental.
The females treated with 50 mg/kg/day and both sexes at 12.5 mg/kg bw/day compared favorably with the control values.
All other parameters were considered to be unaffected by treatment.
The observed effects were reversible during the recovery period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related differences in the urinalysis parameters of males and females at all dose levels. At 50 mg/kg bw/day and 200 mg/kg bw/day, significantly lower pH was recorded in males (p<0.05 and p<0.01, respectively). This was considered to be unrelated to treatment.

There were no late effects during the recovery period.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No findings of toxicological relevance were noted in week 13 in male or female rats in test item-treated groups.
The mean fore- and hind limb grip strength values of the test item-treated rats compared favorably with those of the respective control rats.
A statistically significant differences (increased, p<0.01) in the mean locomotor activity of the males treated with 200 mg/kg bw/day was noted during first measurement interval (0-6 minutes) and in females treated with 50 mg/kg bw/day (decreased, p<0.05) during the third measurement interval (18-24 minutes). The transient nature of these differences (in combination with the absence of a dose response relationship in females) were considered to be indicative of toxicological irrelevance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related differences in organ weights included significantly higher mean relative kidney weights in males at 50 mg/kg bw/day (p<0.05) and 200 mg/kg bw/day (p<0.01), and lower mean absolute thymus weight (p<0.01), mean thymus-to-body weight ratio (not significant) and mean thymus-to-brain weight ratio (p<0.05) in females at 200 mg/kg bw/day.
However, these differences did not correlate with microscopical changes of toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related adverse macroscopical findings at any dose level. A small number of typical background macroscopical findings were noted in males and females of the control and high-dose groups. The observed findings were either also (or only) seen in control animals, or only in individual animals of the high-dose group.

Male no. 22 (12.5 mg/kg bw/day), found dead on day 80 of treatment, was autolytic and no macroscopical findings could be recorded. Female no. 97 (200 mg/kg bw/d) was sacrificed for ethical reasons on day 51 of treatment. No macroscopical findings were evident.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related microscopic findings were recorded in the stomach. At the end of the main study and in unscheduled deaths these findings consisted of:
At the end of the main study and in the two unscheduled deaths treatment related microscopic
findings consisted of:
Stomach
- forestomach erosion at slight degree was noted in one group 4 (200 mg/kg bw/day) male and at minimal degree in one group 4 (200 mg/kg bw/day) female.
- minimal forestomach ulceration was recorded in one group 4 (200 mg/kg bw/day) female
- forestomach squamous hyperplasia at minimal or moderate degree in two group 4 (200 mg/kg bw/day) males and in females at minimal degree in one group 2 (12.5 mg/kg bw/day), two group 3 (50 mg/kg bw/day) and seven group 4 (200 mg/kg bw/day) animals

Following the four week recovery period, minimal forestomach squamous hyperplasia remained in evidence in one female previously treated with 200 mg/kg bw/day. These findings are indicative of local irritation in the forestomach.
The remaining microscopic findings were within the range of background pathology encountered in Wistar rats at these ages in this type of study and occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:

not examined

Details on results:

HISTORICAL CONTROL DATA
Historical control data are provided in the study report for the following parameters: hematology, biochemistry, urinanalysis

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: clinical signs

2

Dose descriptor:

LOEL

Remarks:

local effects

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: forestomach effects (forestomach squamous hyperplasia)

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Analysis of dose formulations

The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R2) exceeded than 0.99.

The PETMP peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no significant peak appeared at the retention time of PETMP and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed..

The PETMP concentrations in the dose formulations ranged from 82.4% to 105.8% with reference to the nominal and were within the accepted range of ±20%. Single results found did not deviate more than 5.9% from the corresponding mean and met the specified acceptance criterion of ≤15%, confirming homogeneous distribution of PETMP in the preparations.

The test item was found to be stable in application formulations when kept four hours at room temperature and eight days at room temperature. Recoveries met the variation limit of 15% from the time-zero (homogeneity) mean value, except for group 2 when kept eight days at room temperature that exceeded the required limit up to 7.3%. In conclusion, the results indicate the accurate preparation of the test item PETMP in vehicle during this study.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, a no-observed-effect level (NOEL) could not be identified due to local effects at the forestomach. A specific target organ was not apparent. The no-observed adverse effect level (NOAEL) was considered to be 50 mg/kg bw/day.

Executive summary:

In a subchronic toxicity study according to OECD Guideline 408 (21 September 1998) and EU Method B.26 (30 September 1996 ), PETMP (97.4% a.i.) was administered to 10 RccHan: WIST(SPF) rats/sex/dose in corn oil by gavage at dose levels of 0, 12.5, 50 and 200 mg a.i./kg bw/day (corrections were made for purity) for 91 or 92 days.Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 28 days without treatment to detect recovery from, or persistence of, toxic effects.

Test item-related findings were noted in some animals on the first day of treatment: tachypnea, convulsions and/or shivering, prostration, hunched posture, ruffled fur and/or stiff gait. Pica, a typical protective behavior for rats, was observed at this dose. These signs were generally seen only on day 1. The findings were of limited incidence and transient, and were considered to indicate acute toxicity from a metabolic overload which triggered a subsequent adaptive) if nonspecific) metabolic response. Findings of toxicological relevance were not seen at 50 mg/kg bw/day or 12.5 mg/kg bw/day.

There were no test item-related deaths, no differences in mean food consumption or body weights, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test item-related differences in the ophthalmoscopy, no test item related effects on hematology or urine parameters. There were no test item-related macroscopic findings.

Differences noted in mean levels of sodium, potassium and chloride of male rats treated with 50 mg/kg bw/day and 200 mg/kg bw/day were considered to be test item-related. Females treated with 200 mg/kg bw/day had higher mean potassium and chloride levels. The potassium levels exceeded the upper range of the historical control data. The females treated with 50 mg/kg bw/day and both sexes at 12.5 mg/kg bw/day compared favorably with the control values. All other parameters were considered to be unaffected by treatment.

Test item-related differences in organ weights included higher mean relative kidney weights in males at 50 mg/kg bw/day and 200 mg/kg bw/day, and lower mean absolute thymus weight, mean thymus-to-body weight ratio and mean thymus-to-brain weight ratio in females at 200 mg/kg bw/day. However, these differences did not correlate with microscopical changes of toxicological relevance.

Test item-related microscopic findings were recorded in the stomach. At the end of the main study and in the two unscheduled deaths, these findings consisted of forestomach erosion (slight in degree) in one male at 200 mg/kg bw/day male and in one female at 200 mg/kg bw/day, forestomach ulceration (minimal in degree) in one female at 200 mg/kg bw/day, and forestomach squamous hyperplasia (minimal or moderate in degree) in two males at 200 mg/kg bw/day. This latter finding (minimal in degree) was also observed in one female at 12.5 mg/kg bw/day, two females at 50 mg/kg/day) and seven females at 200 mg/kg bw/day. Minimal forestomach squamous hyperplasia persisted after the recovery period in one female previously treated with 200 mg/kg bw/day. These findings are indicative of local irritation in the forestomach.

Based on these results, a no-observed-effect level (NOEL) could not be identified, and a specific target organ was not apparent. The LOEL for local effects (forestomach squamous hyperplasia) was 12.5 mg/kg bw/day. The LOEL for local effects, based on local irritation at the site of application (forestomach), is judged as not relevant to humans due to significant different anatomic situation and exposure probability in humans.

The no-observed adverse effect level (NOAEL) relevant to human DNEL calculation was considered to be 50 mg/kg bw/day.

This subchronic toxicity study in the ratis acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rat.