Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
between 16 May 2012 and 20 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol
- Physical state: Liquid
- Expiration date of the lot/batch: 2013-09-05
- Storage condition of test material: Room temperature in the dark
- Other:
Description: Amber coloured liquid
Label: Performance Additive (1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol)

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least five weeks. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks
- Weight at study initiation: males weighed 316 to 357g, the females weighed 193 to 223g
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum (a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK))
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: between 17 July 2012 (first day of treatment) and 31 August 2012 (final necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

PREPARATION OF DOSING SOLUTIONS: The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test material is soluble in arachis oil.
- Concentration in vehicle: 12.5, 62.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were analysed for concentration of the test material by HPLC. The results indicate that the prepared formulations were within ± 4 % of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused: During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable.
- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug and sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
Animals were treated daily for up to seven weeks at the appropriate dose level throughout the study (except for females during parturition where applicable). Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. The male dose groups were killed on Day 43.
Frequency of treatment:
once daily.
Duration of test:
Chronological Sequence of Study:
-Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
-On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
-Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
-Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
-The male dose groups were killed and examined macroscopically on Day 43.
-At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day oral range-finding in rats, where NOAEL of 1000 mg/kg bw was established (Harlan Laboratories, 2012 Project No. 41200906)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

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

During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes (as g/rat/day)

Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily ((with the exception of the pairing phase).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
- Organs examined: All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology:
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10 % formalin, except where stated:
Coagulating gland
Prostate
Epididymides♦
Seminal vesicles
Gross Lesions
Testes♦
Ovaries
Uterus/Cervix
Mammary gland (females only)
Vagina
Pituitary

♦ = preserved in Bouin’s fluid then transferred to 70 % Industrial Methylated Spirits (IMS) approximately forty-eight hours later

All tissues were despatched to the Test Site for processing. The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment related kidney changes, examination was subsequently extended to include similarly prepared sections of kidney from animals from the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

The uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes (according to OECD421 study)
- Skeletal examinations: Yes (according to OECD421 study)
- Head examinations: Yes (according to OECD421 study)

For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post
partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were
calculated retrospectively from this data)
All live offspring were assessed for surface righting reflex on Day 1 post partum.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p < 0.05. Statistical analysis was performed on the following parameters:
 
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
 
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
 
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Indices:
Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre–implantation loss = (number of corpora lutea - number of implantati on sites/number of corpora lutea) x100
% post–implantation loss = (number of implantati on sites - total number of offspring born/number of implantati on sites) x 100

ii) Live Birth and Viability Indices:
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (number of offspring alive on Day 1/number of offspring) x 100
Viability Index (%) = (number of offspring alive on Day 4/number of offspring alive on Day 1) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula: (number of male offspring/total number of offspring) x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: There were no treatment-related effects on mating performance, fertility and gestation length.

Details on maternal toxic effects:
There were no unscheduled deaths, toxicologically significant clinical signs or significant effects detected in body weight development of treated animals. No adverse effect on food consumption, food efficiency or water consumption was detected in treated animals when compared to control animals.There were no treatment-related effects on mating performance and fertility. The gestation lengths of treated females was comparable to controls (between 22 and 23½ days). All males treated with 1000 mg/kg bw/day had mottled kidneys, seven of which also had enlarged and pale kidneys, one male also had enlarged kidneys and one male also had pale kidneys. One female treated with 1000 mg/kg bw/day had pale kidneys. Seven males treated with 250 mg/kg bw/day and one male treated with 50 mg/kg bw/day had mottled kidneys. No such effects were detected in females treated with 50 mg/kg bw/day. One female treated with 250 mg/kg bw/day had a malformed and fluid filled left horn of the uterus which was detached from the cervix. This animal also had no visible vaginal opening. There were no treatment-related effects on absolute or relative organ weights. Statistical analysis of the data did not reveal any significant intergroup differences.

The following treatment related microscopic findings were detected:
Kidneys: Males from all treatment groups showed treatment-related lesions characterized by hyaline droplets accumulation in the cytoplasm of the proximal
convoluted tubules, tubular degeneration and regeneration of the proximal convoluted tubules, granular casts in the inner portion of cortex and medulla, interstitial fibrosis and mixed cell infiltration. The severity of the lesions was dose-proportional. It is likely that the degenerative / regenerative and inflammatory lesions were secondary to the hyaline droplet accumulation in the cytoplasm of the tubular epithelium.
In females treated with 1000 mg/kg bw/day, the nature of the kidneys lesions was different. Tubular vacuolation of the proximal portion was evident in two females and in one of these females which was affected more severely; it was associated to a minimal single cell necrosis of the epithelium.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses:
In total, nine females from the control and 50 mg/kg bw/day dose groups, eight females from the 250 mg/kg bw/day dose group and all females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size:
No significant differences were detected for corpora lutea, implantation counts or litter size for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

VIABILITY (OFFSPRING)

Live birth index was statistically significantly reduced in litters from females treated with 250 mg/kg bw/day. The intergroup difference was considered to be attributable to isolated litters and in the absence of a dose related response was considered not to be of toxicological importance.

CLINICAL SIGNS AND BODY WEIGHT (OFFSPRING)

There were no toxicologically significant effects in group mean values for total litter weights, offspring body weights and body weight changes and surface righting reflex. Statistical analysis of the litter or surface righting reflex data did not reveal any significant intergroup differences.
A statistically significant increase in male offspring weights in litters from females treated with 1000 mg/kg bw/day was evident on Day 1 post partum. An increase in this parameter is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance. No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, weak, no milk in stomach, physical injuries, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

SEXUAL MATURATION (OFFSPRING)

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material related effects.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations

Dose Level (mg/kg bw/day)

0 (Control)

50

250

1000

Mated pairs

n

10

10

9

10

Females showing evidence of copulation

n

10

10

9

10

Pregnant females

n

9

9

8

10

Conception Days 1-5

n

10

10

9

10

Gestation = 22 Days

n

2

3

0

0

Gestation = 22 ½ Days

n

4

2

1

4

Gestation = 23 Days

n

1

1

5

6

Gestation = 23 ½ Days

n

2

3

2

0

Dams with live young born

n

9

9

8

10

Dams with live young at Day 4post partum

n

9

9

8

10

Corpora lutea/dam  

x

14.0

14.1

15.3

14.0

Implants/dam

x

13.5

13.2

14.1

12.4

Live offspring/dam at Day 1post partum

x

11.9

11.7

10.0

11.2

Live offspring/dam at Day 4post partum

x

11.9

11.0

9.1

10.8

Sex ratio: % males at Day 1post partum

x

48.7

51.1

43.6

45.8

Sex ratio: % males at Day 4post partum

x

48.7

51.0

44.1

46.3

Litter weight (g) at Day 1post partum

x

70.74

69.64

58.79

72.31

Litter weight (g) at Day 4post partum

x

104.41

97.30

82.03

99.20

Male offspring weight (g) at Day 1post partum

x

6.08

6.17

6.16

6.68

Male offspring weight (g) at Day 4post partum

x

8.95

9.32

9.47

9.45

Female offspring weight (g) at Day 1post partum

x

5.87

5.80

5.61

6.30

Female offspring weight (g) at Day 4post partum

x

8.72

8.89

8.80

9.08

LOSS OF OFFSPRING/DAM

 

 

 

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

 

 

 

0

n

6

4

2

1

1

n

1

3

4

5

2

n

0

1

1

2

3

n

1

1

1

1

4

n

0

0

0

1

Pre-natal (implantations minus live births)

 

 

 

 

 

0

n

3

2

1

4

1

n

1

1

1

3

2

n

1

5

2

1

3

n

1

1

0

1

4

n

2

0

1

1

6

n

0

0

1

0

8

n

0

0

1

0

10

n

0

0

1

0

Post natal (live births minus offspring alive on Day 4post partum)

 

 

 

 

 

0

n

9

6

3

7

1

n

0

1

3

2

2

n

0

1

2

1

3

n

0

1

0

0

4

n

0

0

0

0

n= Number

x = Mean

Applicant's summary and conclusion

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for neonatal toxicity was 1000 mg/kg bw/day.
Executive summary:

An Oral (gavage) reproduction/developmental toxicity screening test in the rat wasperformed in order to investigate potential adverse effects of 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol on reproduction including offspring development (OECD 421, Harlan Laboratories, 2013). The test item was administered by oral gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 250 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25post coitum. For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964, cited in the study report). All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

There were no unscheduled deaths and toxicologically significant clinical signs detected in treated animals. Body weight development, food and water consumption were unaffected. Regarding reproductive performance, no treatment-related effects were recorded on mating and conception rates. There were no differences in gestation length. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 23 ½ days for each group.

In total, nine females from the control and 50 mg/kg bw/day dose groups, eight females from the 250 mg/kg bw/day dose group and all females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. No significant differences were detected for corpora lutea, implantation counts or litter size for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. Live birth index was statistically significantly reduced in litters from females treated with 250 mg/kg bw/day. The intergroup difference was considered to be attributable to isolated litters and in the absence of a dose related response was considered not to be of toxicological importance. There were no toxicologically significant effects in group mean values for total litter weights, offspring body weights and body weight changes and surface righting reflex. Statistical analysis of the litter or surface righting reflex data did not reveal any significant intergroup differences. A statistically significant increase in male offspring weights in litters from females treated with 1000 mg/kg bw/day was evident on Day 1 post partum. An increase in this parameter is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance. No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, weak, no milk in stomach, physical injuries, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

In adult animals, there were signs of systemic toxicity. At necropsy,males treated with 1000 mg/kg bw/day had mottled kidneys, seven of which also had enlarged and pale kidneys, one male also had enlarged kidneys and one male also had pale kidneys. One female treated with 1000 mg/kg bw/day had pale kidneys. Seven males treated with 250 mg/kg bw/day and one male treated with 50 mg/kg bw/day had mottled kidneys. No such effects were detected in females treated with 250 or 50 mg/kg bw/day.There were no treatment-related effects on absolute or relative organ weights. Statistical analysis of the data did not reveal any significant intergroup differences. At the histopathological evaluation, the following treatment related microscopic abnormalities were detected in the kidneys: males from all treatment groups showed treatment-related lesions characterized by hyaline droplet accumulation in the cytoplasm of the proximal convoluted tubules, tubular degeneration and regeneration of the proximal convoluted tubules, granular casts in the inner portion of cortex and medulla, interstitial fibrosis and mixed cell infiltration. The severity of the lesions was dose-proportional. It is likely that the degenerative / regenerative and inflammatory lesions were secondary to the hyaline droplet accumulation in the cytoplasm of the tubular epithelium. In females treated with 1000 mg/kg bw/day, the nature of the kidneys lesions was different. Tubular vacuolation of the proximal portion was evident in two females and in one of these females which was affected more severely; it was associated with a minimal single cell necrosis of the epithelium.

In conclusion, the oral administration of 1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg bw/day, resulted in treatment related microscopic kidney effects in males from all treatment groups and in females treated with 1000 mg/kg bw/day. No such effects were detected in females treated with 50 or 250 mg/kg bw/day.

Therefore, the “No Observed Effect Level” (NOEL) for systemic toxicity is 250 mg/kg bw/day in females. In males, the kidney effects detected in all treatment groups were considered to represent an adverse effect of the test item, therefore a ‘No Observed Adverse Effect Level’ (NOAEL) has not been established in the male rat. However, the kidney changes of hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons (Alden 1986, Hard, 2008, cited in the study report). This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings consisting of tubular degeneration/regeneration, granulated tubular casts, interstitial fibrosis and mixed cell infiltration in males are more likely to be correlated to the same condition as hyaline droplets and are therefore considered to represent limited relevance to humans. In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant, a NOAEL for males can be established at 1000 mg/kg bw/day. The NOEL for reproductive toxicity and regarding neonatal toxicity was 1000 mg/kg bw/day.