Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
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.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : AS 100 (Di-isopropyl xanthogen polysulphide)
Description : yellow liquid
Batch number : 246041110023
Date received : 05 August 2011
Storage conditions : room temperature in the dark
Expiry date : 18 July 2012

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.


































Effect levels (P0)

open allclose all
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'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
Treatment related:
yes

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.