Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January/February 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol
EC Number:
293-927-7
EC Name:
1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol
Cas Number:
91648-65-6
Molecular formula:
not applicable (UVCB substance)
IUPAC Name:
1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol
Test material form:
not specified
Details on test material:
- Description: yellow-brown liquid
- Storage condition of test material: ambient, dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf, Switzerland
- Age at study initiation: 8 weeks (at acclimatisation)
- Weight at study initiation: Males 154 to 180 g; Females 154 to 172 g (at acclimatisation)
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding (‘Lignocel’, Schill AG, 4132 Muttenz, Switzerland).
- Diet: Pelleted standard Kliba no. 343, Batches 35/88 and 36/88 rat maintenance diet (‘Kliba’, Klingentalmuehle AG, 4303 Kaiseraugst, Switzerland) ad libitum.
- Water: Community tap water from Itingen was available ad libitum via individual cage water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target 22 +/- 3
- Humidity (%): target 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
From 2 January 1989 - 20 February 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle: justification for use of polyethylene glycol was not reported
- Concentration in vehicle: 0, 5, 20 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Approximately 2 g of each test article/vehicle mixture was exactly weighed into a 100 mL volumetric flask. 4 mL of dichloromethane was added, this was then made to volume with acetonitrile. If necessary, these solutions were diluted with acetonitrile to yield concentrations within the calibration range; subsequently, they were analyzed by HPLC.
- HPLC model used: Merck 655A pump, photometer, integrator and sampling unit
- HPLC column: RP Select B (5 µm; 125 x 4.6 mm)
- Mobile Phase: Acetonitrile:bidistilled water (95:5 v/v)
- Detection method: UV detection at 300 nm
- Flow rate: 2 mL/min
- Linearity range: 12.6 - 126 mg/L
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (0 mg/kg): 5 male and 5 female + 5 male and 5 female for 14 day recovery period
Group 2 (50 mg/kg): 5 male and 5 female
Group 3 (200 mg/kg): 5 male and 5 female
Group 4 (1000 mg/kg): 5 male and 5 female + 5 male and 5 female for 14 day recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected based on data received from the sponsor
- Rationale for animal assignment (if not random): computer-generated random algorithm
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per day

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal/day: Yes

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 data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 4 and 6 weeks
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: HB, HCT, MCV, MCH, MCHC, PLATELETS, RETIC., NEN, WBC, Diff. WBC Count, red cell morphology, PT, PTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 4 and 6 weeks
- Animals fasted: Yes (18 h)
- How many animals: all animals
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total

URINALYSIS: Yes
- Time schedule for collection of urine: 4 and 6 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: None
Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data. Univariate one-way analysis of variance was used to assess the significance of intergroup differences. 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 between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher’s exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No mortality an no signs or symptoms which could be related to test article treatment were observed in the animals during the entire treatment or treatment-free recovery period.
Mortality:
no mortality observed
Description (incidence):
No mortality an no signs or symptoms which could be related to test article treatment were observed in the animals during the entire treatment or treatment-free recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
increased body weight without toxicological significance
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
changes in the enzyme levels without toxicological significance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute relative liver and kidney weights of male and female rats were significantly increased.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Slight hepatic parenchymal hypertrophy; renal tubular inclusion bodies,
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No death occurred during the entire treatment and the additional treatment-free recovery period.
No signs or symptoms which could be related to test article treatment were observed in the animals during the entire treatment or treatment-free recovery period.
Due to the vehicle used for test article dilution (polyethylene glycol, PEG 400) all animals showed diarrhoea during the treatment period. This diarrhoea was observed in control animals between days 4 and 28 and in animals of groups 2 to 4 between days 9 and 28 of the treatment phase. This symptom was not observed during the treatment-free recovery period.

BODY WEIGHT AND WEIGHT GAIN
A statistically significant increase in body weights was observed in female rats of the high-dose group 4 when compared to the control animals. This significant increase was seen during the whole treatment as well as during the treatment-free recovery period.
In addition to this observation the females of group 3 (mid-dose) showed an increase in body weights at days 15 and 28 of the treatment period and those of the low-dose group 2 at day 15. These findings were considered to be spontaneous in nature and within normal limits of biological variation. They were of no biological or toxicological relevance.

FOOD CONSUMPTION
No statistically significant differences which could be related to test article treatment were observed throughout the entire treatment or treatment-free recovery period when the food consumption of the control animals was compared to those of the animals of the three test article treated groups.

OPHTHALMOSCOPIC EXAMINATION
No treatment related ophthalmoscopic changes were observed at termination of treatment and treatment-free recovery period.

HAEMATOLOGY
The assessment of haematological data indicated no changes of toxicological significance at the end of the treatment nor at the end of the treatment-free recovery period.
All statistically significant differences noted between control and test article-treated rats were considered to be incidental and of normal biological variation for rats of this strain and age with no biological relevance.

CLINICAL CHEMISTRY
For biochemical data the following effects were noted at termination of the treatment when compared to the respective controls:
— slightly decreased glucose level for males of group 4;
— slightly increased total cholesterol level for males of groups 3 and 4;
— slightly increased gamma-glutamyl-transferase (G-GT) activity for males of group 4;
— slightly increased calcium concentration for males of group 4;
— slightly increased albumin and total protein concentration for both sexes of group 4.
These findings were considered to be of metabolic nature due to an increased metabolic functional load and not of toxicological relevance.
Also noted, was a dose-related decrease in enzyme activity for alanine aminotransferase (ALAT) for both sexes of all treated groups at the end of the treatment. The decrease was slight and considered to be related to an inhibitory effect of the test article or metabolites thereof, with the assay procedure.
For all of the changes noted, the findings were found to be reversible at the end of the treatment-free recovery period and comparable to those of the controls. All other differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

URINALYSIS
Urinalysis data indicated no changes of toxicological significance. The only change noted, was a slight increase in the overnight urine output (volume/18 hours) for both sexes of group 4 at the end of the treatment. This is considered to be related to a greater fluid intake and not an effect related to renal damage. At the end of the treatment-free recovery period this observation was found to be reversible and comparable to that of the controls.

ORGAN WEIGHTS
At terminal sacrifice after the 4 week treatment period the absolute relative liver and kidney weights of male and female rats were significantly increased in group 4 animals. At the same time the kidney weights and ratios were increased in females of group 2 and the ratios in males of group 3. Spleen weights and ratios were increased in females of group 3 and spleen ratios in males of group 4. At sacrifice after the additional 2 week recovery period the absolute and relative liver weights were still increased in female animals of the high-dose group 4.

GROSS PATHOLOGY
No treatment related findings were recorded.

HISTOPATHOLOGY: NON-NEOPLASTIC
Some high dose animals showed slight hepatic parenchymal hypertrophy which was not apparent after the recovery period. Renal tubular inclusion bodies were present in greater numbers in mid and high dose males than in controls. High dose females had some similar inclusions which were not seen in controls. These effects were much reduced or absent after the recovery period. Slight renal tubular degeneration was seen in some main test high dose males but not after the recovery period.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical chemistry and biochemistry parameters are not adverse but adaptive effects; pathology/histopathology parameters (increased kidney weight ratios in male rat) are species specific and not relevant to humans.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Organ Weights (g) Summary (after 4 weeks) MALES

 

Group 1

0 mg/kg

Group 2

50 mg/kg

Group 3

200 mg/kg

Group 4

1000 mg/kg

Body weight

Mean

SD

N

267.8

27.9

5

268.7

4.3

5

251.1

12.2

5

258.7

21.4

5

Pituitary

Mean

SD

N

0.011

0.002

5

0.009

0.001

5

0.010

0.001

5

0.010

0.003

5

Liver

Mean

SD

N

7.91

1.14

5

9.25

0.84

5

8.14

1.11

5

10.19 *

1.22

5

Kidneys

Mean

SD

N

1.94

0.24

5

2.04

0.24

5

2.16

0.31

5

2.48 *

0.28

5

Adrenals

Mean

SD

N

0.066

0.013

5

0.071

0.004

5

0.063

0.013

5

0.067

0.009

5

Spleen

Mean

SD

N

0.515

0.089

5

0.585

0.086

5

0.589

0.092

5

0.652

0.103

5

Testes

Mean

SD

N

2.87

0.08

5

2.97

0.18

5

3.05

0.24

5

2.95

0.20

5

*/** Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 2: Organ Weights (g) Summary (after 4 weeks) FEMALES

 

Group 1

0 mg/kg

Group 2

50 mg/kg

Group 3

200 mg/kg

Group 4

1000 mg/kg

Body weight

Mean

SD

N

200.7

9.2

5

196.0

5.1

5

201.7

8.6

5

202.8

9.4

5

Pituitary

Mean

SD

N

0.013

0.003

5

0.017

0.004

5

0.013

0.001

5

0.015

0.003

5

Liver

Mean

SD

N

6.22

1.00

5

6.77

0.79

5

6.61

0.25

5

8.53 **

0.43

5

Kidneys

Mean

SD

N

1.33

0.07

5

1.54 *

0.08

5

1.44

0.08

5

1.58 **

0.15

5

Adrenals

Mean

SD

N

0.088

0.015

5

0.101

0.020

5

0.102

0.012

5

0.095

0.013

5

Spleen

Mean

SD

N

0.303

0.087

5

0.530

0.046

5

0.678 **

0.073

5

0.583

0.048

5

Ovaries

Mean

SD

N

0.094

0.013

5

0.099

0.006

5

0.107

0.017

5

0.108

0.019

5

*/** Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:
The study report describes a guideline study which was conducted under GLP compliance. Based upon the results obtained, the No-Observed-Adverse-Effect-Level (NOAEL) of the test material is 50 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days. This is based on the findings observed in clinical biochemistry and pathology/histopathology parameters.
Executive summary:

A subacute 28 day toxicity study was performed to assess the repeated dose toxicity of the test material (CAS No. 91648-65-6) in the SPF-bred Wistar rat following the method described in OECD Guideline 407 (Ullmann, 1989). The test substance was administered daily by gavage to 4 groups of ten animals (five males and five females) at concentrations of 0 (control, Group 1), 50 (Group 2), 200 (Group 3) and 1000 (Group 4)mg/kg/day. A further 10 animals (five males and five females) were included in Group 1 and Group 4 as recovery animals. All animals were subjected to daily clinical observation. Body weight and food consumption were measured weekly and on the day before necropsy. During week 4 and week 6 of treatment, both eyes of all animals were examined. During week 4 and week 6 blood and urine was collected from each animal for clinical laboratory investigations and urinalysis. Subsequently, a necropsy examination was performed, macroscopic observations and organ weights were recorded. A histopathological examination was performed on adrenals, heart, kidneys, liver, spleen and stomach.

At 50 mg/kg/day no treatment-related changes were detected. At 200 mg/kg/day and 1000 mg/kg/day several clinical chemistry parameters were either slightly increased (total cholesterol (males, Group 4), gamma-glutamyl-tranferase (males, Group 4), calcium (males, Group 4), albumin (males and females, Group 4) and total protein (males and females, Group 4) or slightly decreased (glucose (males, Group 4). These findings were not considered to be the result of test material administration butwere considered to be of metabolic nature due to an increased metabolic functional load and not of toxicological relevance. There were no treatment related necropsy findings. High-dose animals (Group 4) showed increased absolute and relative liver and kidney weights after the 4-week treatment period. The absolute and relative liver weights of the females of the same group were still increased after the 2-week treatment-free recovery period. The kidney weights and ratios were increased in males from Group 3 and the spleen weights and ratios in females of Group 3 and ratios only in males of Group 4 at the terminal sacrifice after 4 weeks of treatment.The changes in the spleen weights and ratios in females are not biologically relevant as there was no dose-response effect (no effect was seen at the highest dose level 1000 mg/kgIt) and it was not supported by any parameter of clinical laboratory investigation or pathology/histopathology. Some high dose animals showed slight hepatic parenchymal hypertrophy which was not apparent after the recovery period. High and mid dose group animals had increased numbers of renal tubular inclusions which, in some high dose males, were associated with slight tubular degeneration. This phenomenon was much reduced in severity or absent after the recovery period.

From the results presented in this report, a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg/day was established.

Proposal for a relevant NOAEL (28 -day repeated dose toxicity study)

In the following, a justification for setting the NOAEL which is different from the study report is given. The study results have been re-examined and a conclusion was made according to the current state of the science. According to the study report, NOAEL of 50 mg/kg bw was established due to the clinical biochemistry and pathology/histopathology parameters of male and female animals.

The effects observed in animals at dose level of 200 mg/kg bw

Clinical effects observed in the animals in the 28-day study at 200 mg/kg bw are treatment related effects but not adverse (Ullmann et al., 1989; Lewis et al., 2002). They are all adaptive effects because there were no histopathological findings in kidney and spleen, no toxicologically meaningful effects in haematology and in clinical chemistry, no other microscopical lesions etc. The findings at this dose level are summarized in the following table:

Dose

Male

Female

200 mg/kg/day

-      Increased kidney weight ratios;

-      Slightly increased total cholesterol level;

-      Decrease in ALAT activity.

-      Increased spleen weights;

-      Decrease in ALAT activity.

The increase in the male kidney weight ratios is almost certainly due to the hyaline droplets that were discovered in the Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 421, Harlan Laboratories, 2012, Report No. 41200907). According to the study report, the mechanism of hyaline droplets accumulation in the proximal tubules is specific for male rats and is assumed not being toxicologically relevant for humans (Lewis et al., 2002; EPA/25/391/019F; Hard, 2008). This finding is adverse for the male rats but has limited relevance for other species including man. Therefore, this effect is considered to be adaptive in nature. The changes in the spleen weights and ratios in females are not biologically relevant as there was no dose-response effect (no effect was seen at 1000 mg/kg, the highest dose level) and the effect was not seen in the 421 study. In addition, the biochemistry values and histopathology show that effects on the spleen are unlikely.

The only other effects that were seen at 200 mg/kg were an increase in cholesterol levels in the males and a slight decrease in alanine aminotransferase levels for both sexes. However, according to the study report, cholesterol increase was considered to be of metabolic nature due to an increased metabolic functional load and not of toxicological relevance. The slight decrease in enzyme activity for alanine aminotransferase (ALAT) was observed not only in this dose group but was present in all treated groups and in both sexes. “The decrease was slight and considered to be related to an inhibitory effect of the test article or metabolites thereof, with the assay procedure”. Generally, for all of the clinical changes noted, “the findings were found to be reversible at the end of the treatment-free recovery period and comparable to those of the controls. All other differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation for rats of this strain and age”.

 

Conclusion

Due to the fact that effects found in kidneys of males are specific effects for male rats and not relevant for humans, as well as all other effects observed in treated animals at 200 mg/kg bw were adaptive and not adverse, this dose level is considered to be a NOAEL for the purposes of risk assessment.