Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No study required as available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
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

Developmental Toxicity: Han Wistar rats, 20/ group, 0, 25, 75 or 250 mg/kg/day on Day 6 to 19 after mating

, oral (gavage): NOAEL = 250 mg/kg/day (maternal toxicity & embryo-fetal survival and development) (OECD 414, GLP)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
OECD 407
Reason / purpose:
reference to other study
Remarks:
Range-finding study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15-25°C)
- Stability under test conditions: Stability of the formulation was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.
Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 77 to 83 days old
- Weight at study initiation: 177 to 211 g (Day 0 of gestation)
- Fasting period before study: no
- Housing:
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 and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
The cages constituting each group were blocked by group and mounted in batteries.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
Aspen chew block : A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
Number of animals per cage:
Acclimatization: up to four animals
During pairing: one (stock) male and one female
Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water ad libitum from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): not stated, 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: 8 March 2017 (animal arrival) To: 3 to 6 April 2017 (necropsy)
Route of administration:
oral: gavage
Vehicle:
other: 98% Water with 2% Kolliphor EL(cremophor) v/v
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Comparability with previous studies
- Concentration in vehicle: 0, 2.5, 7.5, 25 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Stability was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.

CHROMATOGRAPHIC CONDITIONSCHROMATOGRAPHIC CONDITIONS
High performance liquid chromatograph (HPLC)
Column: Poroshell 120 SB-C18 2.7 µm, 150 × 4.6mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile phase: Acetonitrile/0.05% aqueous formic acid 50/50 v/v
Flow rate: 0.8 mL/min
Rinse solvent: Acetonitrile/water 50/50 v/v
Detector wavelength: UV, 252 nm
Injection volume: 10 µL
Time constant: 1.0 sec
Sampling rate: 2 points/sec
Run time: 8 minutes

These conditions were established using a Waters Alliance 2695 separation module and 2487 detector.
Calibration standard range: 2 µg/mL – 10 µg/mL.
Extraction solvent: Acetonitrile / 0.05% aqueous formic acid 80/20 v/v
Diluent: Acetonitrile / 0.05% aqueous formic acid 50/50 v/v
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: until pregnancy
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. When positive evidence of mating was detected, this day referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Day 6-19 of pregnancy
Frequency of treatment:
daily
Duration of test:
mating to necropsy (day 20 of pregnancy), 22-25 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females, 20 stock males (not part of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 25, 75 and 250 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were chosen based on the results of the preliminary study for effects on embryo-fetal development in the Han Wistar rat by oral gavage administration.
In that study, dose levels of 10, 40 and 160 mg/kg/day were well tolerated with no clinical signs and no effect upon body weight or food consumption. There was no clear effect of maternal treatment upon numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss. Placental, litter and fetal weights and litter size were also all unaffected by treatment.
As there were no clear treatment related findings, a dose level of 250 mg/kg/day was selected for the high dose (250 mg/kg/day was assessed on a 14 day dose range finder study where the main findings were changes in the forestomach, but not deaths). This was to try and elicit a slight treatment related response, as required by the regulatory authorities.
Therefore it was considered that dose levels of 25, 75 and 250 mg/kg/day would be suitable for use on this main study.
- Rationale for animal assignment (if not random): random
- Other: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 recorded daily during the treatment period at the following times in relation to dose administration: One to two hours after completion of dosing, As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: 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.

OTHER: Reproductive Assessment
The following were recorded for all animals:
Uterus: Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn, number of Corpora lutea, Implantation sites, Resorption sites (classified as early or late), Fetuses (live and dead).
Apparently non pregnant animals: The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, E, 1964)].
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter; 50% Sexed internally and eviscerated
- Skeletal examinations: Yes
- Head examinations: Yes:
Statistics:
Due to limitations of this free text field, see "Any other information on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values.
Historical control data:
available
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs observed following dose administration that were considered to be related to treatment with Caprolactam disulfide.
Two females receiving 250 mg/kg/day were observed with slight piloerection on Day 19 of gestation.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean bodyweight gain (Days 6-20) and bodyweight gain on Days 6-7, 12-13 and 15-16 of gestation was slightly but statistically significantly lower for females receiving 250 mg/kg/day when compared with Controls.
Mean bodyweight and bodyweight gain for females receiving 25 or 75 mg/kg/day was similar to that of the Controls and were considered to be unaffected by treatment with Caprolactam disulfide.
Gravid uterine weight was essentially similar across all groups of females, with only females that received 250 mg/kg/day having a slightly but statistically significant lower adjusted maternal bodyweight change (Day 6 20) when compared with Controls (approximately 83% of Controls).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption was considered unaffected by treatment with Caprolactam disulfide during Days 6- 19 of gestation when compared with Controls.
Food intake was slightly but statistically significantly lower during Days 6-9 of gestation for females receiving 250 mg/kg/day when compared with Controls.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females treated with Caprolactam disulfide up to dose levels of 250 mg/kg/day on Day 20 of gestation did not reveal any findings considered to be related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Sex ratio (% male) and pre-implantation loss was statistically significantly higher for females that received 25 mg/kg/day when compared with Controls, however, as the mean percentage of pre implantation loss was within the Historical Control Data range (4.6-11.4%, 4 main embryo-fetal studies, 2016-2017) and there was no dose response, hence these findings were not considered related to treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
none stated
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Pregancy was terminated on Day 20.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregancy was terminated on Day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female receiving 250 mg/kg/day was not pregnant, all other females were pregnant.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
number of live young essentially similar to Controls
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio (% male) and pre-implantation loss was statistically significantly higher for females that received 25 mg/kg/day when compared with Controls, however, as the mean percentage of pre implantation loss was within the Historical Control Data range (4.6-11.4%, 4 main embryo-fetal studies, 2016-2017) and there was no dose response, hence these findings were not considered related to treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
In all groups that received Caprolactam disulfide, there was a slight increased incidence of delayed ossification of cranial centres when compared to Controls, but this incidence was within the Historical Control Data range. A delay in ossification is a transient stage in fetal development and not considered adverse.
At 25 mg/kg/day there was an isolated high incidence of 20 thoracolumbar vertebrae compared to Control. This finding was outside of the Historical Control Data range, but in the absence of any other vertebral configuration abnormalities, this finding is not thought to be adverse or related to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: vertebra
Description (incidence and severity):
At 25 mg/kg/day there was an isolated high incidence of 20 thoracolumbar vertebrae compared to Control. This finding was outside of the Historical Control Data range, but in the absence of any other vertebral configuration abnormalities, this finding is not thought to be adverse or related to treatment.
Key result
Developmental effects observed:
no

see attachments

Conclusions:
The study was perfomed acc. OECD TG 414 under GLP and was sufficiently documented, hence, the results can be considered sufficiently reliable to assess the developmental toxicity of Caprolactam disulfide in rats. No effects related to treatment were observed up to the highest dose tested, 250 mg/kg bw/d, but doses were considered to be well-chosen as other repeated dose toxicity studies revealed i.a. a NOAEL of 40 mg/kg bw/d for general toxicity.
It was concluded from this study that the dosage of 250 mg/kg/day was the maternal no observed-adverse-effect-level (NOAEL) and 250 mg/kg/day was the no observed adverse-effect-level (NOAEL) for embryo-fetal survival and development.
Executive summary:

The purpose of this study acc. OECD TG 414 under GLP was an assessment of the influence of Caprolactam disulfide on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat.

Three groups of 20 females received Caprolactam disulfide at doses of 25, 75 or 250 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 98% water with 2% Kolliphor EL (cremophor) v/v at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

Results

Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation were not adversely affected by treatment with Caprolactam disulfide at doses up to 250 mg/kg/day when compared with Control animals. At 250 mg/kg/day, there was a slight reduction in mean body weight gain during Days 6-7 of gestation and marginal reductions in adjusted body weight gain, and mean food consumption during Days 6-9 of gestation.

Embryo fetal survival, growth and development were unaffected by treatment with Caprolactam disulfide at doses up to 250 mg/kg/day.

Conclusion

It was concluded from this study that the dosage of 250 mg/kg/day was the maternal no‑observed-adverse-effect-level (NOAEL) and 250 mg/kg/day was the no‑observed‑adverse-effect-level (NOAEL) for embryo-fetal survival and development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
high quality (OECD 414 study under GLP)
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

As there were no adverse effects on reproduction or development were noted, no MoAA can be performed, and there is no indication that the observed absence of adverse effects in rats is not relevant for humans as no indication of species-specifity is given.

Justification for classification or non-classification

As there were no adverse effects on reproduction or development were noted, no classification as reproductive toxicant is triggered.