bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to Reproduction (screening): oral: gavage, rat (Sprague-Dawley) m/f, 10/sex/dose, 0, 100, 330, 1000 mg/kg bw/d in corn oil, (OECD TG 422, GLP): no adverse effects in adults or offspring noted, NOAEL = 1000 mg/kg (systemic toxicity), NOAEL = 1000 mg/kg (reproductive toxicity)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-05 - 2018-01-05 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD 422 guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

URCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Refrigerated (2 to 8°C) and protected from the light.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 69 to 76 days old
Females 83 to 90 days old
- Weight at study initiation:
Males 338 to 408 g
Females 239 to 298 g
- Fasting period before study:
no
- Housing:
Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
A soft white untreated aspen wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter was provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.
- Diet (e.g. ad libitum):
SDS VRF1 Certified pelleted diet ad libitum
- Water (e.g. ad libitum):
Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum. Bottles were changed at appropriate intervals
- Acclimation period:
Males: six days prior to the commencement of treatment.
Females: 20 days prior to the commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

ENVIRONMENTAL CONDITIONS
Rodent Facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- Temperature (°C): / Humidity (%):
Temperature and relative humidity were monitored and maintained within the range of 20-24ºC and 40-70%. There were no deviations from these ranges.
- Air changes (per hr):
Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):
Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES:
From: 16 August 2017 (males), 2 August 2017 (females)
To: 26 September 2017 (males), 11 to 15 October 2017 (females) (F0 necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8°C).

- VEHICLE: Corn oil
- Concentration in vehicle: 0, 20, 66, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. These investigations demonstrated that formulations in the 10 to 200 mg/mL concentration range are homogenous and stable following ambient storage (15-25°C) for one day and following refrigerated storage (2-8°C) for 15 days.
Achieved concentration: Samples of each formulation prepared for administration in Week1 of treatment and on Day 10-12 of lactation were analyzed for achieved concentration of the test item.

Homogeneity and Stability in Corn Oil Formulations
The homogeneity and stability of the test item in corn oil formulations were assessed at nominal concentrations of 10 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub divided into 4 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Homogeneity and Stability of Dose Formulations
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Concentration of Dose Formulations
The mean concentrations of the test item in test formulations analyzed during the study. The original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis. In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for The test item in corn oil formulations at nominal concentrations of 10 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The mean concentrations of The test item in test formulations analyzed for the study were within the applied limits of +10/-15% of nominal concentrations, confirming accurate formulation, with the exception of Day 10-12 of lactation Group 3 samples which were 15.8% of nominal concentration.

Duration of treatment / exposure:

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

Frequency of treatment:

Once daily, at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 100, 330 and 1000 mg/kg/day) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories.
In the preliminary study dose levels of 250, 500 and 1000 mg/kg/day were investigated. There were no test item-related premature deaths, no signs observed in relation to dose administration, no test item-related changes in clinical condition, body weight, water consumption or spleen weights. Group mean food intake was slightly reduced during the treatment period when compared with pre-treatment values for all dose groups. Mean absolute and body weight relative liver weights were slightly high for females receiving 1000 mg/kg/day and for males receiving 500 or 1000 mg/kg/day but one male at 250 mg/kg/day had a small liver. Kidney weights also exhibited a modest dose dependent reduction for body weight relative data in males. Macroscopic findings were limited to adhesions involving multiple organs of the thoracic cavity for two animals receiving 250 mg/kg/day which were considered to be related to mis-dosing. In addition, one animal receiving 1000 mg/kg/day was found to have cysts on the kidneys.
Therefore, a high dose level of 1000 mg/kg/day was selected for this study as the limit dose for the OECD 422 guideline. The intermediate and low dose levels of 330 and 100 mg/kg/day were chosen to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).
- Rationale for animal assignment (if not random):
Allocation: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure variations in body weight of animals did not exceed ± 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation:
Irregular estrous cycle: Six females
Unexpected death: One female

Positive control:

not required

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
Clinical and Behavioral Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing: Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 – daily; Week 2 onwards - once each week
F0 females: Week 1 – daily; Week 2 – once, Gestation phase - Days 0, 7, 14 and 20; Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation; One to two hours after completion of dosing; As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see above

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Weekly during acclimatization; Before dosing on the day that treatment commenced (Day 1) and weekly thereafter; On the day of necropsy.
F0 females: Weekly during acclimatization; Before dosing on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows for F0 animals: Weekly before pairing, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Days 15 to 22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: Days 0-6, 7-13 and 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: not stated

OTHER: See related entry of the present OECD 422 in section “Repeated Dose Toxicity”

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs
Wet smears: Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 11 to 14 of lactation).

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

Parturition Observations and Gestation Length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records Made During Littering Phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Thyroid Hormone Analysis: Blood samples were collected as follows:
Day 4 of age: Offspring: two females selected for sampling per litter (if possible):
If four female offspring available - one female was selected for T4 (serum)#
If five or more female offspring available - one female was selected for T4 (serum) and one female for TSH (plasma)
No females were selected for sampling if:
The resultant live litter size would fall below ten pups.
The resultant number of live females would fall to less than three (for subsequent allocation to the Day 13 procedures) e.g. three female offspring in the litter - no offspring selected
Day 13 of age: Offspring: two males and two females per litter (where possible):
two for T4 (serum); where possible one male and one female (Priority was given to serum sample)
two for TSH (plasma); where possible one male and one female

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after week five
- Maternal animals: All surviving animals on day 14 post partum
In Detail:
F0 males: After final investigations completed (after at least four weeks of treatment).
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 14 of lactation (following terminal blood sampling).
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age; Scheduled kill - Day 13 of age.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the table in the free text field were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Selected offspring for thyroid hormone analysis - Day 4 of age. / Scheduled kill - Day 13 of age.
Method of kill:
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.
F1 offspring on Day 4 of age: Blood sampling required.
Externally normal offspring discarded without examination.
Externally abnormal offspring examined, and retained pending possible future examination.
F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination.
Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.

Statistics:

Due to limitations of this free-text field, see "Any other information on materials and methods incl. tables.

Reproductive indices:

Estrous Cycle
The percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating / Animals paired) * 100
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) * 100
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) * 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born / Number pregnant) * 100

Offspring viability indices:

Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) * 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) * 100
Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) * 100
Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) * 100
Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
Percentage males = (Number of males in litter / Total number of offspring in litter) * 100
Group mean values were calculated from individual litter values.

Offspring Examinations
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test item at doses of 100, 330 and 1000 mg/kg/day during the 5 week treatment period for males, and during the 2-week pre-pairing period, gestation and lactation periods for females was well tolerated.
Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing. With the exclusion of the mortality, there were no test item-related signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment.
On Day 1 of lactation Group 3 female (No. 67) receiving 330 mg/kg/day was terminated early due to total litter loss. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Group 4 female (No. 97) receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment: the female was clinically normal before dosing but showed signs of decreased activity, was cold to touch, had thin build, piloerection, partially closed eyelids and whole body pallor after dosing. Macroscopic examination did not reveal any cause for the poor condition of this animal and there was no sign of mis-dosing

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of animals receiving the test item at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment throughout the 5 week dosing period for males and the 2-week pre-pairing period, gestation and lactation periods for females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period with males receiving 330 mg/kg/day affected predominantly in the latter part of the treatment period, only. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.
Mean food consumption for males receiving 100 mg/kg/day and for females receiving 100 or 330 mg/kg/day were generally similar to that of the Controls and was therefore unaffected by the test item administration.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females given 100, 330 or 1000 mg/kg/day, which attained statistical significance at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.
All groups of treated males showed slightly lower than Control reticulocyte concentration, however there was no dose relationship apparent. Among females given 1000 mg/kg/day haemoglobin concentration was slightly lower than Control, with statistical significance attained; this difference was attributable to a single female (No. 83) with an atypical low haemoglobin concentration and no effect of treatment was inferred. In addition, mean cell haemoglobin concentration was slightly low for females at all dose levels with statistical significance being attained for those females receiving 330 or 1000 mg/kg/day, however there was no dose relationship apparent.
All other haematological differences from control observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The sensory reactivity observations conducted during Week 5 of treatment for males and Days 7 to 9 of lactation for females revealed no findings which were considered treatment related.

Motor activity assessment of males during Week 5 of treatment revealed a statistically significant higher incidence of overall high beam scores for animals receiving 1000 mg/kg/day when compared to Controls. Males receiving 100 or 330 mg/kg/day had also shown a slightly higher incidence of overall high beam scores when compared to Controls however, did not attain statistical significance. In addition, males receiving 100, 330 or 1000 mg/kg/day when compared to Controls, have shown a higher overall incidence of low beam scores. Overall low beam scores were statistically significantly higher for males receiving 100 or 1000 mg/kg/day. The increase in motor activity observed in males receiving the test item was considered unrelated to treatment as both overall high and low beam scores lacked a dose-dependent trend and Control performance is lower than in the Historical Control data.
Motor activity assessment of females at Days 7 to 9 of lactation has revealed a slight dose dependent increase in the overall scores for both high and low beam break activity but differences did not attain statistical significance. The higher incidence of high beam scores attained statistical significance for females receiving 1000 mg/kg/day at the 36 minute interval whereby the incidence of low beam scores attained statistical significance at both the 30 and 36 minute intervals. The increase in motor activity observed in the females was not associated with any clinical signs or signs recorded during the arena observation and therefore these differences were considered fortuitous in nature and were unrelated to treatment with the test item.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology
Decedents
Female No. 97 (given 1000 mg/kg/day) was killed for welfare reasons on Day 3 of treatment due to poor clinical condition. At macroscopic examination, the adrenals were dark and the duodenum, jejunum and stomach were distended with green and greasy fluid. Dark colour of the adrenals correlated microscopically with sinusoidal dilatation/congestion. There was also slight hypertrophy of the zona fasciculata of the adrenal cortex. The stomach showed diffuse inflammation of the submucosa/mucosa and erosion of the mucosa of the glandular and non-glandular regions. Apoptosis was present in the cortex of the thymus and mesenteric lymph node.

Microscopic Pathology
Animals Killed After 5 Weeks of Treatment
Treatment Related Findings
Changes related to treatment with the test item were seen in the mesenteric lymph nodes and liver.
Mesenteric Lymph Nodes
Erythryocytosis/erythrophagocytosis (minimal or slight severity) was present in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male.

Summary of treatment related findings in the mesenteric lymph nodes for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Erythrocytosis/Erythrophagocytosis
Minimal 1 0 3 0 0 0 0 1
Slight 0 0 0 4 0 0 0 0
Total 1 0 3 4 0 0 0 1
Cellularity Increased, Follicles
Minimal 1 2 0 5 0 0 0 0
Total 1 2 0 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Liver
Centrilobular hypertrophy was present as a diffuse change in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.
Summary of treatment related findings in the liver for animals killed after 5 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 100 330 1000 0 100 330 1000
Hypertrophy, Centrilobular
Minimal 0 3 5 1 0 0 0 0
Slight 0 0 0 4 0 0 0 0
Total 0 3 5 5 0 0 0 0
Number of tissues examined 5 5 5 5 5 5 5 5

Incidental Findings
Mineralisation and tubular degeneration were present in the tubular epithelium of predominantly proximal convoluted tubules in several females given 1000 mg/kg/day and in controls. The distribution of mineralisation in control and treated females indicates alteration in calcium/phosphorus homeostasis. However, the incidence and severity were similar in control and treated females so the findings are considered incidental and unrelated to treatment.
All other findings were considered incidental and not related to administration of the test item.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with the test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment.
All females were not cycling before termination (Days 11 to 14 of lactation) and were in diestrous at termination.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with the test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment and one female receiving 330 mg/kg/day (3F No. 62) was found to be not pregnant.

Formulation Analysis
The homogeneity and stability of the test item in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 days and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 5%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.
The mean concentrations of the test item in test formulations from the original Week 1 samples were extracted but due to repeated system issues, no results can be reported. The contingency samples were analyzed in replacement of the original samples. These results are all within 4% of nominal concentration, confirming accurate formulation. The coefficient of variation values are within 2%, confirming precise analysis.
In Day 10-12 of lactation (females), the Group 2 and 4 results were within 5% of nominal concentration, and the coefficient of variation values were within 3%, confirming precise analysis. The Group 3 samples were -36.4% of nominal concentration with a coefficient of variation of 58.11%. Excluding the low ‘bottom’ result as an outlier due to the very low result, the Group 3 samples were -15.8% of nominal concentration with a difference from mean of ±12.56%. The Group 3 contingency samples were analyzed outside of the confirmed stability period, and therefore can only be reported for information purposes only. These contingency results were -8.5% from nominal concentration with a coefficient of variation of 5.59%.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

haematology

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: no adverse effects at the highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One Group 3 female (No. 67) receiving 330 mg/kg/day was terminated early on Day 1 of lactation due to a total litter loss.
The mean number of implantation sites and litter size were unaffected by parental treatment at all dose levels, with all litter size values within range of the Historical Control Data. The mean number of implantations for Controls is at the higher range of the Historical Control Data.
The post implantation survival index was unaffected by parental treatment with the test item.
Offspring survival to Day 13 of age and sex ratio appear unaffected by parental treatment up to and including 1000 mg/kg/day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 13 of age the mean body weights of male and female offspring receiving 1000 mg/kg/day were slightly lower than that of the Control. As this difference was slight, did not attain statistical significance and there was no apparent dose response, it was considered that there was no effect on mean offspring body weights of parental treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring Ano-genital Distance: For males receiving 330 or 1000 mg/kg/day, the mean and adjusted mean values for ano genital distance were slightly higher than that of Controls. This slight difference was minimal and as there were no effects observed on the male reproductive system, it is considered this finding was not adverse. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.
Offspring Nipple Count: A check was performed to assess for the presence of nipple/areolae for the male offspring.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Offspring Macropathology: There were no findings in the decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See above
Offspring Ano-genital Distance: For males receiving 330 or 1000 mg/kg/day, the mean and adjusted mean values for ano genital distance were slightly higher than that of Controls. This slight difference was minimal and as there were no effects observed on the male reproductive system, it is considered this finding was not adverse. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.
Offspring Nipple Count: A check was performed to assess for the presence of nipple/areolae for the male offspring.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: no adverse effects at the highest dose tested

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Conclusions:

The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential reproductive toxicity (and repeated dose toxicity) of the test item.
Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 13 of lactation was well tolerated.
There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of treatment after dose administration and one female given 330 mg/kg/day was terminated early as result of a total litter loss. Microscopic changes observed for the female killed for welfare reasons included distension of the gastrointestinal tract and inflammation/erosion in the glandular and non-glandular regions of the stomach. The cause of these lesions could not be determined from pathological examination, however there was no clear evidence in those animals surviving to terminal kill of local gastrointestinal toxicity and therefore it was considered that both deaths were unrelated to treatment.
Test item related histopathological changes are likely to be adaptive and non-adverse.
Test item-related microscopic changes were observed in the mesenteric lymph nodes of males given 1000 mg/kg/day,and are considered non-adverse.
Estrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment with The test item at all dose levels investigated, only one female receiving 330 mg/kg/day was found not to be pregnant.
The assessment of endocrine disruptor relevant endpoints comprised the measurement of circulating thyroxine levels (T4) in adult males and in Day 13 offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Body weight adjusted mean values for ano-genital distance were slightly higher for males given 330 or 1000 mg/kg/day than that of Controls. As there were no findings in the other endocrine disruptor endpoints and there were no changes observed in the male reproductive system it is considered this change is not treatment-related.
There was no effect of parental treatment on the mean number of uterine implantations, litter size, sex ratio, offspring birth weights and subsequent body weight gain or on offspring development up to Day 13 of age.
All Control females on study showed regular oestrus cycles during treatment, mated and became pregnant with a live litter. The litter size and offspring survival indices in the Control group were not adversely affected by the mineralisation with mean values within or marginally above those in the Historical Control Data range. All of these observations support the power of the study to detect test item related effects.
In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor. No necessity for classification as reproductive toxicant is given.

Executive summary:

The purpose of this OECD 422 study under GLP was to assess the general systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item (a vulcanisation agent for industrial use) by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration at a volume dose of 5 mL/kg/day. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

During the study, for adult animals assessments of clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Blood samples were collected from all adult animals at termination and from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis.

 

Results

Parental (F0) responses

Oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for two weeks prior to pairing, during pairing and then up to termination of the males after 5 weeks of treatment and females on Day 14 of lactation was well tolerated. 

There were two premature deaths, one female receiving 1000 mg/kg/day was killed for welfare reasons after dosing on Day 3 of treatment and one female receiving 330 mg/kg/day was terminated early due to total litter loss on Day 1 of lactation. Macropathology findings included abnormal pale coloration of the mammary gland and it was noted the gland was inactive. Both premature deaths were considered unrelated to treatment.

With the exclusion of the mortality, there were no test item-related signs observed during the detailed physical examination and arena observations, no post-dosing observations, no effects on sensory reactivity, grip strength and no effects on body weight performance. There was considered to be no effect on motor activity.

Mean food consumption for males receiving the test item at 330 or 1000 mg/kg/day was marginally higher than Controls during the treatment period. The mean food intake of females at 1000 mg/kg/day was slightly higher during the second week of the pre-pairing treatment period, throughout gestation and lactation when compared to Controls.

Estrous cyclicity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment with The test item. One female receiving 1000 mg/kg/day was found to be acyclic during treatment and one female receiving 330 mg/kg/day (3F No. 62) was found to be not pregnant.

The haematological examinations at scheduled termination revealed, when compared with Controls, a dose-dependent reduction in activated partial thromboplastin time in females at all dose levels. Activated partial thromboplastin time for males receiving the test item, however, were generally similar to Control values.

Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males receiving 1000 mg/kg/day where alkaline phosphatase was lower compared to Controls and both alanine aminotransferase and aspartate aminotransferase concentration were higher compared to Controls. A dose dependent response was observed for alanine aminotransferase and aspartate aminotransferase only. Liver enzyme changes were observed in females with slightly higher alanine aminotransferase (330 or 1000 mg/kg/day only) and aspartate aminotransferase concentrations compared to Controls. Unlike the males however, these values were in the absence of both a dose dependent response and statistical significance.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring.

Changes in organ weights consisted of slightly high body weight adjusted mean liver weights for males at 1000 mg/kg/day when compared to Control.  

Macroscopic examination of the adult males revealed abnormal dark colour of the mesenteric lymph node in four males that received 1000 mg/kg/day and observed in one male at dose levels of 100 and 330 mg/kg/day. 

Histopathological evaluation of retained tissues revealed erythryocytosis/erythrophagocytosis in the sinuses of the mesenteric lymph nodes of several males given 330 or 1000 mg/kg/day, one female given 1000 mg/kg/day and one control male. Increased cellularity of the follicles (minimal severity) was present in the mesenteric lymph nodes of all males given 1000 mg/kg/day, two males given 100 mg/kg/day and one control male. Centrilobular hypertrophy was present in the liver of most males given 100 mg/kg/day and all males given 330 or 1000 mg/kg/day.

 

F1 litter responses

The clinical condition, litter size, sex ratio, body weight and survival of the F1 offspring was unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment. One female given 330 mg/kg/day had a total litter loss. Mean and adjusted mean values for ano-genital distance were marginally higher for males given 330 or 1000 mg/kg/day compared to the Controls. The mean ano-genital distance of female offspring was essentially similar to Controls in all treated groups.

 

Conclusion

In conclusion, within the context of this combined repeated dose toxicity and reproductive/ developmental toxicity screening study it was concluded that a dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity and reproductive/developmental toxicity in the CD rat. The test item showed no evidence of being an endocrine disruptor.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study was conducted on the registered substance itself acc. OECD TG 422 under GLP. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

See above, Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422),no adverse effects in adults or offspring noted.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on lack of any adverse effects on reproductive toxicity (and general systemic toxicity), it is rather impossible to hypothesize a concrete mode of action.

Estrous cycle length, mating performance, fertility, reproductive capacity and gestation length were unaffected by treatment with the test item at all dose levels investigated, only one female receiving 330 mg/kg/day was found not to be pregnant. 

The assessment of endocrine disruptor relevant endpoints comprised the measurement of circulating thyroxine levels (T4) in adult males and in Day 13 offspring, measurement of the ano-genital distance of offspring on Day 1 of age and nipple counts for male offspring on Day 13 of age. Body weight adjusted mean values for ano-genital distance were slightly higher for males given 330 or 1000 mg/kg/day than that of Controls. As there were no findings in the other endocrine disruptor endpoints and there were no changes observed in the male reproductive system it is considered this change is not treatment-related.

There was no effect of parental treatment on the mean number of uterine implantations, litter size, sex ratio, offspring birth weights and subsequent body weight gain or on offspring development up to Day 13of age.

All Control females on study showed regular oestrus cycles during treatment, mated and became pregnant with a live litter. The litter size and offspring survival indices in the Control group were not adversely affected by the mineralisation with mean values within or marginally above those in the Historical Control Data range. All of these observations support the power of the study to detect test item related effects.

Summarizing, the oral (gavage) administration of the test item to Sprague-Dawley rats, at dose levels of 100, 330 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. So, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day as well as the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the Repeated dose / reproduction toxicity screening test on rats, the no observed adverse effect level (NOAEL) of the test item was determined. The oral (gavage) administration of the test item to Sprague-Dawley rats, at dose levels of 100, 330 or 1000 mg/kg bw/day did not produce any adverse effects in adults or offspring. So, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day as well as the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

No effects on fertility were noted up to the limit dose of 1000 mg/kg, and no systemic toxicity was noted. Consequently the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.