Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2017 to 06 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Purity: >90%
- Description: Brown viscous liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Housing
Animals were housed in a limited access room in cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid polycarbonate bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week. Grid bottomed polypropylene cages that were suspended above absorbent paper, which was changed daily, were used during pairing. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Temperature and relative humidity were monitored and maintained within the range of 20-24ºC and 40-70%. The air supply was filtered fresh air, which was not recirculated. Artificial lighting was set to provide 12 hours light and 12 hours dark.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The Crl:CD(SD) rat strain was used because it is recognized by regulatory agencies as an appropriate model for conditions of possible human exposure and because of the historical control data available at the testing laboratory. The oral gavage route of administration, using a suitably graduated syringe and a rubber catheter inserted via the mouth, at doses of 0, 100, 330 and 1000 mg/kg/day in arachis oil BP, with a dose volume of 5 ml/kg, was chosen based upon results of a 14-day dose range finding oral gavagel toxicity study in the Crl:CD(SD) rat in which there were no mortalities and few signs of toxicity at the high dose of 1000 mg/kg bw.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
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:
The mean concentrations of the test item in dose test formulations analyzed for the study were within ±12% of nominal concentrations, confirming accuracy of the dose formulations.
Duration of treatment / exposure:
Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Frequency of treatment:
Once daily
Details on study schedule:
Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day in arachis oil by oral gavage administration. 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, arachis oil, at the same volume dose as the treated groups.

During the study the following observations were made for the adult animals: clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, serum thyroxine (T4) analysis, oestrous 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 recorded. The serum T4 analysis was performed for the Day 13 offspring. Nipple counts were performed on male offspring on Day 13 of age.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
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
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Envigo, 2018 (range-finding)).

Examinations

Parental animals: Observations and examinations:
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
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):
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:
- 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.
Postmortem examinations (parental animals):
Necropsy
- F0 males: After final investigations completed (after at least five weeks of treatment)
- F0 females failing to produce a viable litter: Day 25 after mating
- F0 females Day 14 of lactation (following terminal blood sampling)

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
- Testes: Initially in modified Davidson’s fluid.
- Eyes: In Davidson’s fluid.

Histology
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Full List:
>All adult animals killed prematurely.
>At scheduled termination, the five lowest numbered surviving F0 males and females with a surviving litter in Groups 1 and 4.
- Abnormalities: All F0 animals.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified.

Light Microscopy
Tissues preserved for examination were examined as follows:
- Premature deaths: F0 males and females from Groups 1 and 4 only.
- Scheduled kill: The first five F0 males and first five lactating F0 females in Groups 1 and 4 and All F0 animals from all groups.
Postmortem examinations (offspring):
SACRIFICE
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled Kill: Day 13 of age.

GROSS NECROPSY and HISTOPATHOLOGY
> Blood samples were collected as follows:
- Day 4 of age: F1 offspring, two females per litter
- Day 13 of age: F1 offspring, two males and two females per litter (where possible).
- All surviving F0 adult males and females (except the female which failed to litter) at termination.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant. For some parameters, including estrous cycles before treatment, stage of estrous cycle at termination and gestation index the similarity of the data was such that analyses were not considered to be necessary. Statistical analyses were carried out separately for males and females using the individual animal as the experimental unit for litter/foetal findings, the litter was taken as the treated unit.
Reproductive indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Group mean values were calculated from individual litter values.
Offspring viability indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 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) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
One non-pregnant, non-litter producing female receiving 330 mg/kg/day was sacrificed on gestation day 25, as specified in the study plan.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For females treated at 1000 mg/kg/day, mean food consumption was slight high when compared with controls prior to pairing (Weeks 1-2) but these variations were marginal and considered incidental.

During gestation, food consumption was comparable to controls in females treated at 100, 330 or 1000 mg/kg/day.

During Days 4-7 and 7 -13 of lactation, mean food consumption was slightly high in females treated at 330 or 1000 mg/kg/day when compared with controls but these variations were marginal with no statistical significance and considered incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation in males of the 330 or 1000 mg/kg/day groups revealed statistically significant decreases of mean prothrombin time (PT) and activated partial prothrombin time (APTT) compared to the concurrent control group.

In addition, for males of the 1000 mg/kg/day group, mean large unstained cells (LUC) and platelet counts were marginally higher than the concurrent control group. These differences were within the historical control data ranges and considered normal biological variations, not treatment related.

Hematological evaluation in females revealed no treatment-related effects.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Potassium concentration in males treated at 100, 330 or 1000 mg/kg/day were slightly high when compared with controls and phosphorus concentration in males and females treated at 100, 330 or 1000 mg/kg/day were also slightly high however these differences were slight and there was no evidence of a dose response.

Furthermore, these changes were within the histrorical control data ranges and are considered incidental.

Triglycerides were low in females treated at 100, 330 or 1000 mg/kg/day however, these values were within the HCD ranges and considered incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were unaffected by treatment.

Group mean high and low beam activity scores for all groups of treated males were low compared with Controls, with statistical significance attained at the 12-minute interval (100 mg/kg/day; high and low beams), the 42-minute interval (1000 mg/kg/day; low beam only) and the total scores (all treated groups, high beams only). However, the majority of these scores were within the historical control data ranges, including some of the scores that have attained statistical significance.

The majority of group mean high and low beam activity scores for all groups of treated females were slightly high compared with Controls with an isolated statistical significance attained at the 42-minute interval for low beams but the differences in the total scores did not attain statistical significance and no effect of treatment is inferred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean absolute and adjusted organ weights in males were unaffected by treatment.

Group mean adjusted heart weights were marginally high in females treated at 100, 330 or 1000 mg/kg/day attaining statistical significance at all dose levels however these weights lacked dose relationship and were within the histrorical control data ranges as such are not considered to be attributable to treatment.

A decrease in thymus weights were observed in females treated at 100, 330 or 1000 mg/kg/day with the difference from controls demonstrating a dose-relationship; these weights did not attain statistical significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed in males after 5 weeks of treatment or in females on Day 14 of lactation revealed no test item-related lesions.

A kidney mass was observed on one male given 1000 mg/kg/day however this was an isolated incidence and was considered to be unrelated to treatment.

Pale areas were seen in the kidneys of several females, including in control animals; there was no relationship to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination revealed accumulations of macrophages which were present in the mesenteric lymph nodes of males and females of animals given 330 or 1000 mg/kg/day.

Vacuolation was present in the adrenal cortex (zona reticularis and zona fasciculata) of two males given 1000 mg/kg/day; as this was only present in two males and the severity of this finding was slight this finding could not be directly attributed to treatment.

A single incidence of nephroblastoma was observed in a male given 1000 mg/kg/day. These tumours do occasionally occur as spontaneous lesions in younger animals such as this in short term studies (Ikezaki et al, 2011). Given the short term nature of this study and the absence of similar lesions in other animals, it is considered that this is an incidental finding and not related to administration of the test item.

Mineralisation was present in the tubules of the renal cortex in females from all groups, including controls. Similar renal changes were not identified in males. The finding is not considered to be related to treatment.

Foci of mucosal congestion and epithelial necrosis were present in the glandular region of the stomach in females from all groups, including controls. These changes are not considered to be related to treatment. The cause of the lesions is unclear.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

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:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Reproductive performance, fertility and offspring survival were unaffected by parental treatment. The No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity and for reproductive/developmental effects was considered to be 1000 mg/kg/day.
Executive summary:

The study was designed to assess the general systemic toxic potential in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral gavage administration for at least five weeks. The study has been performed to the standardized guidelines OECD 422, under GLP conditions.

 

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day in arachis oil by oral gavage administration. 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, arachis oil, at the same volume dose as the treated groups.

 

During the study the following observations were made for the adult animals: clinical observations, body weight, food consumption, hematology (peripheral blood), blood chemistry, serum thyroxine (T4) 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 recorded. The serum T4 analysis was performed for the Day 13 offspring. Nipple counts were performed on male offspring on Day 13 of age.

 

There were no treatment related deaths and no adverse effects on clinical condition, sensory reactivity, grip strength, motor activity, body weight gain, food intake, haematology, and blood chemistry measurements in males and females. In addition, the reproductive endpoints assessed which were unaffected by treatment included estrus cycles, pre-coital interval, gestation length, mating performance, fertility, litter size, offspring survival or offspring sex ratio. 

Offspring clinical condition, body weight, body weight gain, ano-genital distance and external development were also unaffected by treatment. 

 

There was notreatment relatedeffect on the serum T4 levels in all analysed samples from adult male rats and offspring on Day 13 of age.

Group mean absolute and adjusted organ weights in males were unaffected by treatment. A trend of decrease in thymus weights was observed in females treated at 100, 330 or 1000 mg/kg/day with the difference from controls demonstrating a dose-relationship, but the decrease in thymus weights did not attain statistical significance and was considered as non-adverse.

The gross pathology examination performed on males after 5 weeks of treatment or females on Day 14 of lactation revealed no test item-related lesions.

The histopathology examination of a full list of tissues revealed one change that was considered treatment related but non-adverse: accumulations of macrophages in the mesenteric lymph nodes of males and females given 330 or 1000 mg/kg/day.

In conclusion, 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 five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated with no adverse effect observed.

 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size or the growth of the offspring.

 

In the context of this study, the test item showed no evidence of being an endocrine disruptor.

The No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity and for reproductive/developmental effects was considered to be 1000 mg/kg/day.