Registration Dossier

Administrative data

Description of key information

There were no deaths attributable to the test material administration in the 28-day repeated dose toxicity study, 14-day range-finding study and in the Oral (gavage) reproduction/developmental toxicity screening test in the rat (OECD 421). No clinical signs, effects on body weight and food consumption were noted.  Generally, the systemic effects observed in the sub-chronic study and in the Screening Test were similar and confined to the tubular degeneration in males due to the hyaline droplets accumulation, the effect which is adverse for the male rats only and is not relevant to humans. The tubular degenration was also detected in the female animals of the highest dose group (1000 mg/kg bw) in the Screening Test. Therefore, the systemic NOAEL of 250 mg/kg bw established in this study for females (the most sensitive sex in this study) was taken for the purposes of DNEL derivation and risk assessment. Similarly, NOAEL of 200 mg/kg bw was considered appropriate in the 28-day study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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
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
Species:
rat
Strain:
Wistar
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)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral 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.
Duration of treatment / exposure:
daily for up to seven weeks (pregnant females were allowed to give birth and maintain their offspring until day 5 post partum. The malles were killed on day 43).
Frequency of treatment:
The test item was administered daily.
Remarks:
Doses / Concentrations:
50, 250 and 1000 mg/kg bw
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 oral range-finding in rats, where NOAEL of 1000 mg/kg bw was established (Harlan Laboratories, 2012 Project No. 41200906)
Positive control:
No
Observations and examinations performed and frequency:
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).

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Reproductive and developmental parameters (see section 7.8.1)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
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: Yes
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.
Other examinations:
Reproductive performance and litter observations (see section 7.8.1)
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).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
revealed mottled kidneys, enlarged kidneys and/or pale kidneys in males (all dose groups) and
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hyaline droplets accumulation in proximal 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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY

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 WEIGHT GAIN

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.

FOOD CONSUMPTION AND COMPOUND INTAKE

No adverse effect on food consumption or food efficiency was detected in treated animals when compared to control animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

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.

ORGAN WEIGHTS

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

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.

HISTOPATHOLOGY: NON-NEOPLASTIC

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.

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: kidney effects at the highest dose level.
Critical effects observed:
not specified

Table 1.        Incidence and Mean Severity Grade of Kidneys Findings
  Males Females

Group

1 2 3 4 1 2 3 4

Animals and Tissues Examined

10 10 10 10 10 10 10 10

Tub.Degen/Regenerat.

0 6 10 10 0 0 0 0

Mean Severity

0 1.2 2.2 3.5 0 0 0 0

Mixed Cell Infiltration

1 0 8 10 1 0 0 2

Mean Severity

1.0 0 1.8 2.6 1.0 0 0 1.0

Interst. Fibrosis

0 0 7 10 0 1 0 0

Mean Severity

0 0 1.6 2.5 0 1.0 0 0

Tubular Basophilia

1 1 0 0 3 0 1 3

Mean Severity

1.0 1.0 0 0 1.0 0 1.0 1.3

Hyaline Droplets

1 10 10 10 0 0 0 0

Mean Severity

1.0 1.6 2.7 3.6 0 0 0 0

Granular Casts

0 0 10 10 0 0 0 0

Mean Severity

0 0 1.9 2.8 0 0 0 0

Hyaline Casts

0 0 0 0 0 0 0 1

Mean Severity

0 0 0 0 0 0 0 2.0

Tubular Vacuolation

0 0 0 0 0 0 0 2

Mean Severity

0 0 0 0 0 0 0 3.5

Tub. Single Cell Necrosis

0 0 0 0 0 0 0 1

Mean Severity

0 0 0 0 0 0 0 1.0

Grading:

Grade 1 = Minimal Grade

2 = Slight Grade

3 = Moderate

Grade 4 = Marked

No treatment-related effects were detected in the reproductive organs examined.

Conclusions:
The NOAEL of 250 mg/kg bw was established for females. Excluding species specific effects (kidney degeneration as the result of hyaline droplets accumulation in tubulis) in males, which are not relevant for humans, the systemic NOAEL for males is 1000 mg/kg bw.
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 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 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Three studies are available: 28-day oral study in rats, 14-day range-finding study and the Oral (gavage) reproduction/developmental toxicity screening test in the rat (OECD 421). The overal quality of the database is therefore high.

Additional information

14 -day range-finding toxicity study in rats

In a subacute toxicity study, which served as a range-finding study for repeated dose/reproduction screening toxicity testing, ‘1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-nonanethiol’ was administered orally by gavage to each three male and female Wistar Han™:RccHan™:WIST strain rats per dose, for fourteen consecutive days, at dose levels of 50, 250 and 1000 mg/kg bw/day (Harlan Laboratories, 2012, Project No. 41200906). A control group of three males and three females was dosed with vehicle alone (Arachis oil BP).All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing, up to thirty minutes after dosing, one hour and whenever possible 5 hours after dosing. Individual body weights were recorded on Days 1, 4, 8, 11 and 15, food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15, food conversion efficiency was calculated retrospectively, and water intake was measured and recorded daily for each cage group. Necropsy included internal and external macroscopic examination.

There were no compound-related effects in mortality, body weight, food consumption and necropsy: There were no unscheduled deaths, no adverse effects on body weight gains and overall food consumption compared to control were detected as well as no macroscopic abnormalities were noticed in any treated animal.Animals of either sex treated with 1000 mg/kg bw/day showed increased salivation post dosing on Day 14. One female from this treatment group also showed increased salivation post dosing on Days 10 and 13. No such effects were detected in animals of either sex treated with 250 or 50 mg/kg bw/day. Females treated with 1000 mg/kg bw/day showed an increase in overall water consumption when compared to controls. No such effect was detected in males treated with 1000 mg/kg bw/day or animals of either sex treated with 250 or 50 mg/kg bw/day. These observations however are not considered as toxicologically significant effects in treated animals. Hence, no LOAEL could be determined, and the 'No Observed Effect Level' (NOEL) was considered to be 1000 mg/kg bw/day. This subacute toxicity study in the rat is acceptable and satisfies the requirements for a subacute oral range-finding study for repeated dose/reproduction screening toxicity testing in rats.

Oral (gavage) reproduction/developmental toxicity screening test in the rat (OECD 421)

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 on Day 43. Adult females were killed 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 (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. 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. All indices were comparable to controls.The litter parameters were all comparable to controls as well (see section "Toxicity to reproduction").

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 the test material 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. NOAEL of 250 mg/kg bw established for females (the most sensitive sex in this study) was taken for the DNEL derivation.

28 -day oral study in rats

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 but were 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.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study with the longest duration available

Justification for classification or non-classification

No systemic toxicity to organs was observed in three repeated oral studies in rats: the 14 -day range-finding study (Harlan Laboratories, 2012), Oral (Gavage) Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 421; Harlan Laboratories, 2013) and the 28 -day study (Ullmann, 1989). Excluding the degenerative effects in kidneys of male rats which are species specific and are not relevant to humans, no further target organs were affected. The target substance did not cause other relevant significant toxicological effects after repeated oral exposure. Therefore, it does not meet the criteria for classification and will not require labelling for STOT-RE, according to the European regulation (EC) No. 1272/2008.