Registration Dossier

Administrative data

Endpoint:
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:
other: OECD 422 Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat
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)
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.
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.
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.
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
Duration of test:
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.
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 in which there were no mortalities and few signs of toxicity at the high dose of 1000 mg/kg/bw (please see RSS section 7.5.1 Supporting study, Envigo, 2018 (range-finding)).

Examinations

Maternal examinations:
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes (left and right)
Examinations included:
- Number of corpora lutea: No data
- Number of implantations: Yes. Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Each uterine horn For F0 females, the number of uterine implantation sites were recorded.
Fetal examinations:
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content.

- F1 offspring on Day 1 of age:
> Ano-genital distance.

- 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.
> Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.
> Nipple/areolae count, male offspring.
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.
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.
Historical control data:
Historical Control Data (HCD) was presented in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
One female receiving 300 mg/kg/day was sacrificed on gestation day 25. The female was not pregnant and did not produce any litters.
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 historical 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, 300 or 1000 mg/kg/day attaining statistical significance at all dose levels however these weights lacked dose relationship and were within the historical 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

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
clinical biochemistry
behaviour (functional findings)
gross pathology
histopathology: non-neoplastic
maternal abnormalities
pre and post implantation loss
effects on pregnancy duration
dead fetuses
changes in pregnancy duration
changes in number of pregnant

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined

Effect levels (fetuses)

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:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
There was no effect of treatment on the number of implantations, litter size or the growth of the offspring. The No-observed-adverse-effect-level (NOAEL) of the test item 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.

 

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 no treatment related effect on the serum T4 levels in all analysed samples from adult male rats and offspring on Day 13 of age.

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.