Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 27 September 2011 and 08 May 2012. The in-life phase of the study was conducted between 27 September 2011 (first day of treatment) and 10 November 2011 (final necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
An exercise in range finding to determine an NOAEL level for both parental and first generation offspring.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for six days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 304 to 348 g, the females weighed 198 to 218 g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
During the mating 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.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used.
Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperatures and relative humidity controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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 (in a 90-day repeat dose study). Results showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of AS 100 (Di-isopropyl xanthogen polysulphide).

The results indicate that the prepared formulations were within 5± % of the nominal concentration.

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.
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 taken and analysed for concentration of AS 100 (Di-isopropyl xanthogen polysulphide).

The concentration of AS100 Di-isopropyl xanthogen polysulphide in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicate that the prepared formulations were within 5± % of the nominal concentration.

The full method used for analysis of formulations and the results obtained are given in Appendix 17: Chemical Analysis of Test Item Formulations, Methods and Results (see attached background material).
Duration of treatment / exposure:
The test item was administered for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
The test item was administered daily.
Details on study schedule:
Chronological Sequence of Study
i) 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.
ii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii) 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.
iv) 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.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Remarks:
Doses / Concentrations:
control (0 mg/kg bw/day), 10 mg/kg bw/day, 50 mg/kg bw/day, 250 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 males and 10 females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.
Positive control:
No positive control.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
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 (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT:
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:
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).

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:
Water intake was measured daily throughout the study (with the exception of the pairing phase).

REPRODUCTIVE 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 were carried 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):
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.
Sperm parameters (parental animals):
The epididymides and testes were removed from terminal kill adult males and organ weight 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 sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone 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 (Salewski 1964).

All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
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
Ovaries
Testes
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina

All tissues were despatched to the Test Site Harlan Laboratories Ltd., Zelgliweg 1, CH-4452 Itingen, Switzerland for processing. The tissues from control and 250 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 250 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

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.
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 (%) = Numbero 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 implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation 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 born) 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 Day 1 and 4 post partum, using the following formula:

Sex Ratio (% males) = (Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see details on results for discussion of results
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
see details on results for discussion of results
ADULT RESPONSES

MORTALITY:
There were no unscheduled deaths.

CLINICAL OBSERVATIONS:
Clinical findings were confined to transient episodes of increased salivation observed shortly after dosing in test animals from the first or second weeks of treatment in 250 mg/kg bw/day and 50 mg/kg bw/day respectively; persisting sporadically thereafter in both treatment groups through to study termination.

Increased salivation was also observed in three 10 mg/kg bw/day males on Day 31 only. Given the isolated nature of this finding it was considered on this occasion to be related to the gavage procedure and not a result of administration of the test item. No clinical signs were detected 10 mg/kg bw/day females.

One female from the 250 mg/kg bw/day dose group developed generalised fur loss for a short duration but, in isolation this was considered to be incidental.

BODY WEIGHT:
Body weight gain in 250 mg/kg bw/day males throughout the treatment period compared unfavourably with the concurrent control; achieving statistical significance (p<0.01) at Week 1, 3 and 5. A similar trend was evident in females from this dose group with statistical significance achieved during the first week of maturation (p<0.01), and during the second and final weeks of gestation (p<0.05 or p<0.01) and lactation (p<0.05).

Females treated at 50 mg/kg bw/day also showed low weight gains (p<0.05) after one week of treatment with recovery thereafter. No such effects were detected in 50 mg/kg bw/day males or in either sex treated with 10 mg/kg bw/day.

FOOD CONSUMPTION:
Dietary intake in either sex at 250 mg/kg bw/day was generally lower than that of the controls most noticeably in both sexes following the first week of treatment and in females during the lactation phase (p<0.05). Food efficiency (the ratio of body weight gain to dietary intake) in both sexes during the maturation phase and thereafter in males compared unfavorably with that of controls.

No adverse effect on food consumption or food efficiency was detected in either sex treated at 10 and 50 mg/kg bw/day.

WATER CONSUMPTION:
Gravimetric measurement of water intake recorded throughout the treatment period revealed males at 250 mg/kg bw/day to be drinking on average 33% more than the controls and females at 250 mg/kg bw/day to be consuming on average 57% more than the concurrent control.

Water consumption in animals dosed at 10 and 50 mg/kg bw/day remained similar to that of the controls throughout the treatment period.

REPRODUCTIVE PERFORMANCE:
MATING:
There was no convincing evidence of an adverse effect of treatment on mating performance at any of the dosages investigated.

Although, statistical significance (p<0.05) was achieved in 250 mg/kg bw/day females there were no convincing indication of a treatment related effect on distribution of pre-coital intervals for treated animals in comparison with controls evidenced by all animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity).

FERTILITY:
No treatment-related effects were detected on fertility for treated animals when compared to controls. One female treated with 50 mg/kg bw/day and two females treated with 250 mg/kg bw/day did not achieve pregnancy. In the absence of any histopathological correlates in the reproductive organs to elucidate the cause of the nonpregnancy in these animals the intergroup difference was considered to be incidental and of no toxicological importance.

GESTATION LENGTH:
Statistical significance (p<0.01) was achieved in 250 mg/kg bw/day females although this was a likely result of the majority of control females littering early rather than delayed gestation in high dose animals. On this basis, it was considered there were no convincing treatment-related effects detected for the length of gestation between control and treated groups with gestation ranging between 22 ½ and 24 days for control and test animals.

PATHOLOGY:
NECROPSY:
Adults: No macroscopic abnormalities were detected in any test or control animals at study termination.

ORGAN WEIGHTS:
Males at 250 mg/kg bw/day, showed a reduction in absolute and body weight relative epididymides weight in comparison with control values; the differences from control attaining statistical significance (p<0.05). However, mean organ weights were within the historical control range and in the absence of any evidence of histopathological change this finding was considered to be incidental and of no toxicological importance.

There were no other differences in male organ weights detected at dosages of 10 and 50 mg/kg bw/day.

HISTOPATHOLOGY:
There was no evidence of treatment-related histopathological changes in the organs and tissues examined.


































Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Description (incidence and severity):
see details on results for discussion of results
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see details on results for discussion of results
Histopathological findings:
not examined
LITTER RESPONSES:
All 10 and 50 mg/kg bw/day females that produced litters and five of eight females from the 250 mg/kg bw/day test group successfully reared young to Day 5 of age. The litters of three 250 mg/kg bw/day females did not survive beyond Day 4 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, SEX RATIO AND VIABILITY:
No significant differences were detected for corpora lutea, implantation counts for treated animals when compared to controls.

No significant differences were detected for litter size or litter viability at 10 and 50 mg/kg bw/day when compared to controls. Statistical analysis of the litter data from 250 mg/kg bw/day females revealed significant differences in litter size (p<0.001) and litter viability (p<0.01). These latter intergroup differences were considered to be attributable to poor nurturing by the dams rather than a direct effect of 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.

OFFSRPING GROWTH AND DEVELOPMENT:
There was no obvious adverse effect on body weight of the offspring on Day 1 and Day 4 or on body weight gain to Day 4 of age in offspring derived from females at 10 and 50 mg/kg bw/day. However, body weight of female offspring from 250 mg/kg bw/day females achieved significance (p<0.05) on Day 4 and both sexes of offspring from this treatment group showed statistical significance for weight gain to Day 4 of age (males, p<0.05, females p<0.01).

The type and incidence of clinical signs and the performance of the offspring during assessment of surface righting at Day 1 of age did not indicate any obvious adverse effect of treatment at any of the dosages investigated.

At 250 mg/kg/day the group incidence of clinical signs appeared slightly higher than the control and other dosages.

PATHOLOGY:
NECROPSY:
Offspring: Neither the type, incidence nor distribution of macroscopic findings observed for decedent offspring or offspring killed at termination indicated any adverse effect of treatment that could be associated with toxicity of the test item. The higher incidence of premature litter losses at 250 mg/kg bw/day were considered probably to be associated with a lack of maternal attention by the dams at this dosage.

Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: No effect on viability of offspring
Reproductive effects observed:
no
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
Treatment related:
yes

DISCUSSION:

The oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats for a period of up to eight weeks (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of up to 250 mg/kg bw/day resulted in treatment related effects detected in animals of either sex treated with 250 and 50 mg/kg bw/day.

There were no clinical signs to indicate toxicity, however, oral administration of the formulated test item to animals at 250 and 50 mg/kg bw/day induced persistent episodes of transient excessive salivation around the time of dosing. Associated with this finding in high dose (250 mg/kg bw/day) animals was a disproportionate increase in water intake (25% ~ 50%) in comparison with control animals. Excessive salivation and increased water consumption are findings frequently reported following oral administration of slightly irritant/unpalatable test item formulations, and in the absence of supporting toxicity are seldom directly associated with systemic toxicity. High dose animals also showed treatment-related changes for bodyweight gain, dietary intake and utilisation.These changes were also likely to have been associated with the irritative properties of the test item (a hypothesis supported by the findings in a 90 -day repeat dose toxicity study, 41102779).

Further treatment-related changes involved a clear dose-related response in the litters of females receiving 250 mg/kg bw/day in which the numbers of pups, growth and viability (survival rates) were all compromised. However, in the absence of histopathological findings in the reproductive organs examined and in consideration of the treatment-related changes suspected of being associated with the irritant characteristics of the test item it is a reasonable assertion that the cumulative effects of treatment at 250 mg/kg bw/day were sufficient to have prompted stress related responses in the high dose females and this may have been a factor in these animals producing smaller litters and showing a deficit of attention to their offspring (the litters of three of these females did not survive to Day 5 post partum). However, as the reproductive contrast between 250 mg/kg bw/day and controls was clearly discernible a dose-related response in reproduction was clearly identified in this study. Therefore, the possibility that this was a result of reproductive toxicity cannot be ignored. The reproductive performance in females at 10 and 50 mg/kg bw/day were considered to be unaffected by treatment.

Conclusions:
The oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats by gavage, at dose levels of 10, 50 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 50 and 250 mg/kg bw/day. However, given the findings in animals at 50 mg/kg bw/day were confined to minor changes considered to be associated with the administration of a slightly irritant test item the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 50 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined of 10 and 50 mg/kg bw/day animals therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 50 mg/kg bw/day.
Executive summary:

Introduction.

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 10, 50 and 250 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, offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, followed by the termination of all females and surviving offspring on Day 5 post partum. 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.

Results.

Adult Responses:

Mortality. There were no unscheduled deaths.

Clinical Observations. Episodes of increased salivation developed in animals of either sex from the 250 and 50 mg/kg bw/day test groups within the first two weeks of treatment and persisted sporadically thereafter through to study termination.

No treatment-related clinical signs were detected in animals of either sex at 10 mg/kg bw/day.

Body Weight. Impaired body weight development was evident in 250 mg/kg bw/day animals of either sex throughout the study. Females treated at 50 mg/kg bw/day showed low weight gains following one week of treatment with recovery thereafter.

Body weight development in 50 mg/kg bw/day males and in either sex treated with 10 mg/kg bw/day were similar to that of the controls throughout the study.

Food Consumption and Food Efficiency. Dietary intake in either sex at 250 mg/kg bw/day was generally below control values achieving statistical significance in females during the lactation phase. Food efficiency in both sexes during the maturation phase and in males thereafter compared unfavourably with that of controls.

No adverse effect on food consumption or food efficiency was detected in either sex treated at 10 and 50 mg/kg bw/day.

Water Consumption. Gravimetric measurement throughout the study showed 250 mg/kg bw/day animals of either sex to be consuming considerably more water than the corresponding controls. Water intake in animals from the remaining treatment groups remained unaffected by treatment.

Reproductive Performance:

Mating. There were no treatment-related effects on mating for treated animals.

Fertility. There were no statistically significant treatment-related effects on conception rates for treated animals.

Gestation Lengths. There was no convincing evidence of a treatment-related difference in gestation lengths between test and control animals.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. Noteworthy changes were confined to offspring from females treated at 250 mg/kg bw/day. These changes involved statistically significant reductions in litter size and viability when compared to controls.

Offspring Growth and Development. Noteworthy changes were confined to offspring from females treated at 250 mg/kg bw/day. These changes involved statistically significant reductions in body weight gain and litter weights

Offspring Observations. No clinically observable signs of toxicity were detected for offspring from any treatment groups.

Pathology:

Necropsy. Macroscopic findings for adults and offspring did not indicate any adverse effect of treatment.

Organ Weights. No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.

Histopathology. There was no evidence of treatment-related histopathological changes in the organs and tissues examined.

Conclusion.

The oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats by gavage, at dose levels of 10, 50 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 50 and 250 mg/kg bw/day. However, given the findings in animals at 50 mg/kg bw/day were confined to minor changes considered to be associated with the administration of a slightly irritant test item the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 50 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined of 10 and 50 mg/kg bw/day animals therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 50 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has been conducted according to OECD Guideline 421 and GLP and is adequately reported. The study has been assigned a reliability 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats for a period of up to eight weeks (including two weeks pre-mating, gestation and early lactation period for females) at dose levels of up to 250 mg/kg bw/day resulted in treatment related effects detected in animals of either sex treated with 250 and 50 mg/kg bw/day.

There were no clinical signs to indicate toxicity, however, oral administration of the formulated test item to animals at 250 and 50 mg/kg bw/day induced persistent episodes of transient excessive salivation around the time of dosing. Associated with this finding in high dose (250 mg/kg bw/day) animals was a disproportionate increase in water intake (25% ~ 50%) in comparison with control animals. Excessive salivation and increased water consumption are findings frequently reported following oral administration of slightly irritant/unpalatable test item formulations, and in the absence of supporting toxicity are seldom directly associated with systemic toxicity. High dose animals also showed treatment-related changes for bodyweight gain, dietary intake and utilisation.These changes were also likely to have been associated with the irritative properties of the test item (a hypothesis supported by the findings in a 90 -day repeat dose toxicity study, 41102779).

Further treatment-related changes involved a clear dose-related response in the litters of females receiving 250 mg/kg bw/day in which the numbers of pups, growth and viability (survival rates) were all compromised. However, in the absence of histopathological findings in the reproductive organs examined and in consideration of the treatment-related changes suspected of being associated with the irritant characteristics of the test item it is a reasonable assertion that the cumulative effects of treatment at 250 mg/kg bw/day were sufficient to have prompted stress related responses in the high dose females and this may have been a factor in these animals producing smaller litters and showing a deficit of attention to their offspring (the litters of three of these females did not survive to Day 5 post partum). However, as the reproductive contrast between 250 mg/kg bw/day and controls was clearly discernible a dose-related response in reproduction was clearly identified in this study. Therefore, the possibility that this was a result of reproductive toxicity cannot be ignored. The reproductive performance in females at 10 and 50 mg/kg bw/day were considered to be unaffected by treatment.

Reproductive Performance:

Mating.

There were no treatment-related effects on mating for treated animals.

Fertility.

There were no statistically significant treatment-related effects on conception rates for treated animals.

Gestation Lengths.

There was no convincing evidence of a treatment-related difference in gestation lengths between test and control animals.


Short description of key information:
No treatment related effects were detected in the reproductive parameters examined of 10 and 50 mg/kg bw/day animals therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 50 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The OECD 421 reproduction/developmental toxicity screening study is assigned as reliability study 1 and is the only reproductive toxicity study available.

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Echa Approval Provided. Study period May 2015- JUne 2015
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species and strain: Hannover Wistar rats (CRLHan)
Source: Charles River Laboratories, Research Models and
Services, Germany GmbH, Sandhofer Weg 7, D-
97633, from SPF colony
Justification of strain: The rat is regarded as a suitable rodent species for
reproduction studies and the test guideline states it is
the preferred rodent species. The Hannover Wistar rat
was selected due to experience with this strain in
teratology studies.

Animal health: Only healthy animals were used for the test, as certified
by the staff Veterinarian

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.2-22.8°C (target: 22 ± 3 °C)
Relative humidity: 34-66 % (target: 30-70%)
Ventilation: 15-20 air exchanges/hour
Route of administration:
oral: gavage
Details on exposure:
A constant volume of 4 mL/kg bw was administered to all dose groups, including the
controls. The individual volume of the treatment was based on the most recent
individual body weight of the animals.
The control or test item dose formulations were administered to mated, sperm positive
assumed pregnant female rats daily by oral gavage on a 7 days/week basis,
approximately at similar times, from Gestation Day (GD) 6 to GD19, according to the
following dosing scheme:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the AS100 in the vehicle (peanut oil) was assessed in the conditions
employed on the study during the validation study (according to CiToxLAB study
code 14/497-901AN). According to the results, the test item is stable in vehicle in
concentration range of 5 mg/mL - 100 mg/mL for 14 days when stored refrigerated (2-
8ºC).
Analysis of test item formulations for concentration and homogeneity was performed
in the Test Site (Fumoprep Ltd.) using a quantitative IR spectroscopic method (Test
Site study code: FPBSTUDY-121-VAL1, Ref. 4). Duplicate samples (top, middle and
bottom samples) were taken from the test item formulations twice during the study
(during the first and last weeks of treatment). Similarly, one sample (duplicate) was
taken on each occasion from the Group 1 (control) solution for concentration
measurements.
Details on mating procedure:
The oestrus cycle of female animals was examined shortly before start of pairing.
After acclimation, the females were paired according to their oestrus cycle with males
in the morning for approximately 2 hours (1 male: 1 females) until at least 24 sperm
positive females/group are attained. After the daily mating period, a vaginal smear
was prepared and stained with 1% aqueous methylene blue solution. The smear was
examined with a light microscope; the presence of a vaginal plug or sperm in the
vaginal smear was considered as evidence of copulation (GD0). Sperm positive
females were separated and caged individually
Duration of treatment / exposure:
The dams (one control and 3-treated
groups) were treated daily by oral (gavage) administration, from gestation day GD6 up
to and including GD19 (sperm positive day = 0 day of pregnancy, GD0). Caesarean
sections, necropsy of dams and examination of uterine contents were performed on
GD20. The control animals were treated with vehicle only ( peanut oil)
Frequency of treatment:
Daily treatment GD 6 to GD19
Duration of test:
Dosing scheme
Acclimatisation period 5 days- no dosing
Gestation days 5 to 19
Caesarean section and necroscopy Gestation day 20

Remarks:
Doses / Concentrations:
23mg/kg/bw/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
70mg/kg/day
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
210 mg/kg/day
Basis:
analytical conc.
No. of animals per sex per dose:
26-27 females per dose and control group
The number of confirmed pregnant, evaluated dams in the dose groups treated was 26
in the control group and 25 at treated groups.
Control animals:
yes, concurrent vehicle
Details on study design:
accord tp OECD 414
Maternal examinations:
Maternal Data:
- Number of animals at test start, no. of animals surviving, no. of pregnant animals,
no. of animals with total intrauterine mortality
- Clinical signs (by gestation day)
- Mortality (by gestation day), if any
- Body weight and body weight gain: mean ± S.D.
- Corrected body weight on GD20 (body weight-gravid uterine weight) and
corrected body weight gain: mean ± S.D.
- Net body weight change (Body weight on GD20 minus body weight on GD6
minus gravid uterine weight): mean ± S.D.
- Food consumption: mean ± S.D.
- Gross pathology findings
- Gravid uterine weight
- Absolute and relative to body weight liver and spleen weights
Ovaries and uterine content:
Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percent of live foetuses: mean ± S.D.
- Number and percent of intrauterine mortality: mean ± S.D.
Classified according to time of death: Pre-implantation loss, Post-implantation
mortality, Early and late embryonic, as well as foetal death
- Pre-implantation loss: %, group mean
UNumber of corpora lutea-Number of implantationsU x100
Number of corpora lutea
- Post-implantation loss: %, group mean
UNumber of implantations-Number of live foetusesU x100
Number of implantations
Fetal examinations:
Sex distribution: %, group mean
UNumber of male (female) foetusesU x100
Number of foetuses
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
- Visceral abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
- Skeletal abnormalities/litter: %, group mean
UNumber of foetuses with abnormalityU x100
Number of foetuses
Statistics:
Appropriate statistical method (Bartlett,
ANOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, Chi2) using the
litter as the unit for data analysis. The homogeneity of variance between groups was
checked by Bartlett`s homogeneity of variance test. Where no significant
heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If
the obtained result was significant Duncan’s Multiple Range test was used to assess
the significance of inter-group differences. Significant results with inter-group
comparisons were further compared using Kruskal-Wallis, and Mann-Whitney Utests.
Indices:
Pre-implantation loss: %, group mean
UNumber of corpora lutea-Number of implantationsU x100
Number of corpora lutea
- Post-implantation loss: %, group mean
Number of implantations-Number of live foetuses/ Number of implantations x 100
Historical control data:
Hisrorical control data was used to provide clear indications where toxicologically significant effects had occurred
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs: Upon immediate administration of the test material to all treated groups was noted as a dose response effect. Clinical signs included piloerection, clonic convulsion, rooting in bedding and increased salivation.
Body Weight: At 23mg/kg/bw the dams had comparable body weights to control group. There was a marked initial body weight loss noted in mid and high dose groups with partial recovery over the timecourse. The corrected body weight ( adjusted for gravid uterine weight) for both mid and high dose was lower than the control attaining statistical signifcant result.
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
none
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
210 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Mortality : There was no unscheduled mortality in the study.

Clinical signs: Upon immediate administration of the test material to all treated groups was noted as a dose response effect. Clinical signs included piloerection, clonic convulsion, rooting in bedding and increased salivation.

Body Weight: At 23mg/kg/bw the dams had comparable body weights to control group. There was a marked initial body weight loss noted in mid and high dose groups with partial recovery over the timecourse. The corrected body weight ( adjusted for gravid uterine weight) for both mid and high dose was lower than the control attaining statistical signifcant result.

The weight of liver and spleen were increased in mid and high dose groups but no statistically significant changes were seen in liver or spleen weight in low dose group.

Foetal Body weights: Mean foetal body weights were unaffected by maternal treament with test substance at any dose level. The incidence of body weight retarded foetuses was simliar in control and test item treated groups. The weight of foetuses at 23, 70 and 210 mg/kg bw/day did not differ significantly from the control mean, when evaluated by both litter mean and group mean.

Although the overall mean foetal body weights, and the mean weight of foetuses evaluated by litter mean, were statistically significantly higher at the low and mid dose levels (by 5.5 and 6.1%, respectively; all values were in the normal historical range and there was no clear dose response relationship. Therefore these statistical differences were considered not related to treatment.

Teratogenic/Embryotoxic effects: No foetal external abnormalities to the test item at all dose levels up to and including 210mg/kg/day.

No foetal visceral abnormalities are ascribed to the test item administration.

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. The pre-implantation loss was at control level in all treated groups. The total intrauterine mortality was comparable with the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in foetal death compared to the control.

Conclusions:
At both mid and high dose groups maternal toxicity was observed in bodyweight loss and increased liver and spleen weights, however, there was no evidence of adverse treatment related effects seen at external, visceral or skeletal foetal examination at all dose groups.
Executive summary:

This developmental toxicity study was performed to assess the effects of the test item AS100 on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy.

At both mid and high dose groups maternal toxicity was observed in bodyweight loss and increased liver and spleen weights, however, there was no evidence of adverse treatment effects seen at external, visceral or skeletal foetal examination at all dose groups.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
210 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has been conducted according to OECD Guideline 414 and GLP and is adequately reported. The study has been assigned a reliability 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.

In the Prenatal study, Mean foetal body weights were unaffected by maternal treatment with AS100 at any dose level. Although the overall mean foetal body weights, and the mean weight of foetuses evaluated by litter mean, were statistically significantly higher at the low and mid dose levels (by 5.5 and 6.1%, respectively ) all values were in the normal historical range and there was no clear dose response relationship. Therefore these statistical differences were considered not related to treatment.

The mean number of viable foetuses was comparable with the control mean. The sex distribution of foetuses did not differ significantly between the control and treatment groups both for mean and absolute numbers.

Offspring Observations.

No clinically observable signs of toxicity were detected for offspring from any treatment groups.


Justification for selection of Effect on developmental toxicity: via oral route:
The OECD 414 prenatal developmental toxicity study is assigned as reliability study 1 and complements the screening study for fertility and development toxicity study available, OECD 421. The NOAEL for embyrotoxic, foetotoxic and teratogenic effects is over 210mg/kg/day (the high dose group)

Toxicity to reproduction: other studies

Additional information

Historical Findings

The results of a OECD 421 Oral Reproduction/Developmental Toxicity Screening Test in the Rat have been assessed to see if classification as a reproductive toxicant should be applied to the substance.

The oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats by gavage, at dose levels of 10, 50 and 250 mg/kg bw/day showed that no treatment related effects were detected in the reproductive parameters examined of 10 and 50 mg/kg bw/day animals therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 50 mg/kg bw/day.

Treatment-related changes involved a clear dose-related response in the litters of females receiving 250 mg/kg bw/day in which the numbers of pups, growth and viability (survival rates) were all compromised. However, in the absence of histopathological findings in the reproductive organs examined and in consideration of the treatment-related changes suspected of being associated with the irritant characteristics of the test item it is a reasonable assertion that the cumulative effects of treatment at 250 mg/kg bw/day were sufficient to have prompted stress related responses in the high dose females and this may have been a factor in these animals producing smaller litters and showing a deficit of attention to their offspring (the litters of three of these females did not survive to Day 5 post partum). However, as the reproductive contrast between 250 mg/kg bw/day and controls was clearly discernible a dose-related response in reproduction was clearly identified in this study. Therefore, the possibility that this was a result of reproductive toxicity cannot be ignored. The reproductive performance in females at 10 and 50 mg/kg bw/day were considered to be unaffected by treatment.

Based on these results from the OECD 421 study, the effects on the litters at the 250 mg/kg bw/day dose group could be assessed to be caused by cumulative effects of treatment leading to stress in the maternal animals. However, reproductive toxicity cannot be discounted as a dose-related response was observed.

OECD 414 Study on trganogenisis & prenatal development

As seen in the OECD 421, treatment related effects were seen in dams immediately upon dosing with test item. Clinical signs of increased hypersalivation were seen in a dose dependant manner in both studies. The OECD 414 supports the hypothesis that the clinical signs seen were indicative of an irritant property of the test item and the dams stress response may have been the cause of the subsequent poor outcome of the pups in the litters of the high dose group.

In the Prenatal study, Mean foetal body weights were unaffected by maternal treatment with AS100 at any dose level. Although the overall mean foetal body weights, and the mean weight of foetuses evaluated by litter mean, were statistically significantly higher at the low and mid dose levels (by 5.5 and 6.1%, respectively ) all values were in the normal historical range and there was no clear dose response relationship. Therefore these statistical differences were considered not related to treatment.

OECD 414: Teratogenic/Embryotoxic effects: No foetal external abnormalities to the test item at all dose levels up to and including 210mg/kg/day.

No foetal visceral abnormalities are ascribed to the test item administration.

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. The pre-implantation loss was at control level in all treated groups. The total intrauterine mortality was comparable with the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no statistically significant difference in foetal death compared to the control.

Justification for classification or non-classification

Adverse effects on sexual function and fertility

OECD 421 showed no treatment related effects on mating, conception rates, gestation lengths at 10, 50 or 250mg/kg/day.

No treatment related histopathological changes in reproductive organs & tissues examined. A NOEL for systemic toxicity was given at 50mg/kg/day

Advsere effects on development of the offspring

OECD 414 provides data to support non classification. The NOAEL for embyrotoxic, fetotoxic and teratogenic effects was above the highest dose group of 210mg/kg/day though maternal toxicity was observed at LOAEL 70mg/kg/day