Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 16 May 2012 and 20 December 2012. The in-life phase of the study was conducted between 17 July 2012 (first day of treatment) and 31 August 2012 (final necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well-documented GLP Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
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:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
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

Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
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.
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:
The test item was administered daily.
Details on study schedule:
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.
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day range-finding toxicity study in rats (Harlan Laboratories, 2012; Project No. 41200906).
The dose levels were chosen based on the results of previous toxicity work. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study are believed to be of value in predicting the likely toxicity of the test item to man and to screen for potential adverse effects on reproduction.
Positive control:
No

Examinations

Parental animals: Observations and 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).


OTHER:
Reproduction Performance
Mating:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations werecarried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Not examined.
Litter observations:
Litter Data:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

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)

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology:
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 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post 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.

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.
Postmortem examinations (offspring):
Pathology:
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
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).
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (number of animals mated/number of animals paired) x 100
Pregnancy Index (%) = (number of pregnant females/number of animals mated) x 100

Gestation and Parturition Data:
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (number of females delivering live offspring/number of pregnant females) x 100
Offspring viability 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

There were no unscheduled deaths. There were no toxicologically significant clinical signs detected in treated animals. Animals of either sex treated with 1000 and 250 mg/kg bw/day and males treated with 50 mg/kg bw/day showed episodes of increased salivation from Day 11 onwards. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation is considered not to be of toxicological importance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

There were no toxicologically significant effects detected in body weight development.
Males treated with 1000 mg/kg bw/day showed a statistically significant increase in body weight gain during the final week of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.
No toxicologically significant effect on water consumption was detected.
Males treated with 1000 and 250 mg/kg bw/day showed an increase in overall water consumption compared to control animals. Females treated with 1000 mg/kg bw/day also showed an increase in water consumption throughout maturation and gestation. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation the intergroup differences were considered not to be of toxicological importance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Mating:
There were no treatment-related effects on mating performance.

Fertility:
No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female, one female treated with 50 mg/kg bw/day and one female treated with 250 mg/kg bw/day were non-pregnant. No microscopic abnormalities were detected in these females or in the control or 50 mg/kg bw/day male partners. The male partner of the female treated with 250 mg/kg bw/day had moderate inflammation of the prostate which might have affected the mating capabilities of this animal however the inflammation was unrelated to the treatment with the test item. One female treated with 250 mg/kg bw/day was not mated due to the animal having no visible vaginal opening.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. Gestation lengths were between 22 and 23 ½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)

There were no treatment-related effects on absolute or relative organ weights. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS)

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.

HISTOPATHOLOGY (PARENTAL ANIMALS)

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 (P0)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were detected in the reproductive parameters examined.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
reduced in the mid dose group but not in the highest dose group
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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 (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material related effects.

Overall reproductive toxicity

Reproductive 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:
No treatment related effects were detected in the reproductive parameters examined. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity when administered orally by gavage to the rat is 1000 mg/kg bw/day. 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 was performed 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 5 post 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 25 post 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. The following parameters of litters born were comparable to controls: litter size, body weight gain and litter weights and sex ratio at birth and subsequently on Days 1 and 4 post partum. The surface righting reflex of the live offspring (Day 1 post partum was also comparable to controls and no clinically observable signs of toxicity were detected.

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.