Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2017 to 04 June 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
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
EC Number: 948-020-7
Specific details on test material used for the study:
- Purity: > 99% (UVCB)
- Description: Amber liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
Duration of acclimatization:
- Males: six days prior to the commencement of treatment
- Females: 20 days prior to the commencement of treatment

Age of the animals at the start of treatment:
- Males ranged from 69 to 76 days old
- Females ranged from 83 to 90 days old

Weight range of the animals at the start of treatment:
- Males 326 to 386 g
- Females 232 to 295 g

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, 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.

- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24ºC
- Humidity: 40 to 70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Lighting: 12 hours light : 12 hours dark

Animal Replacement:
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 treatment:
- Irregular estrous cycle: Two females

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The required amount of test item was weighed into a suitable container and mixed, by magnetic stirring, with approximately 50% of the final volume of vehicle. Further amounts of vehicle were added and mixed to achieve the required volume. The formulation was magnetically stirred until visibly homogenous.

A series of formulations at the required concentrations were prepared in ascending order by dilution of individual weighings of the test item.

The frequency of preparation was weekly.
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 300 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. 15 days stability was confirmed when stored refrigerated (2 to 8°C) or 1 day when stored at room temperature (15 to 25°C).

The mean concentrations of the test material in formulations analyzed for the study were within applied limits +10/-15% for both GC and HPLC, confirming accurate formulation.
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 at approximately the same time each day.
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, 2017 (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 of all groups
As late as possible in the working day
Ovaries and uterine content:
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.
Female whose litter died before Day 13 of lactation: Mammary tissue appearance.
Fetal examinations:
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

F1 offspring on Day 4 of age:
- Blood sampling
- 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 (but not including those selected for thyroid hormone analysis) 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 one male and one female in each litter.

Animals selected for thyroid hormone analysis: externally normal offspring were discarded without examination. Externally abnormal offspring were examined.
Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including gestation index and stage of estous cycle at termination the similarity of the data was such that analyses were not considered to be necessary.
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:
effects observed, treatment-related
Description (incidence and severity):
On Day 15 of the treatment period excessive chewing, piloerection and partially closed eyelids were evident in all females receiving 1000 mg/kg/day after the dosing procedure.
Mortality:
no mortality observed
Description (incidence):
Five adult females (1 each in the low and mid dose groups and 3 in the high dose group) were sacrificed early (4 out of 5 on post-natal day 1 or 2) during the course of the study due to pup loss according to protocol.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean bodyweight gain for females receiving 1000 mg/kg/day during Week 2 of study was reduced when compared to Controls, and this was reflected in the overall Week 0 to 2 bodyweight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Female animals receiving 1000 mg/kg/day consumed slightly less than Control in Week 1 of treatment (92% of Control).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Both males and females receiving 1000 mg/kg/day had visually consumed more water than the other treated groups and Control.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean absolute and body weight adjusted spleen weights were higher than Control in female animals receiving 1000 mg/kg/day
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dark livers were seen in females sacrificed early (three in the 1000 mg/kg/day group and one in the 330 mg/kg/day group).

Dark spleens were seen in females treated at 1000 mg/kg/day, two females treated at 330 mg/kg/day and one female treated at 100 mg/kg/day.

Dark kidneys were seen in females sacrificed early (3 in the 1000 mg/kg/day group and one in the 330 mg/kg/day group).

The incidence and distribution of all other findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The microscopic examination of F0 adult animals revealed changes related to treatment with the test item in the liver, kidneys, spleen, thyroid and adrenal, but none of these changes was considered adverse.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The post implantation survival index, live birth index and viability index for litters from females receiving 1000 mg/kg/day were lower than Control.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Excluding the clinical signs seen in the pups from the total litter losses, there was a higher incidence of offspring recorded as ‘cold to touch’ in the 1000 mg/kg/day group.
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:
330 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: general systemic toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on mean body weights of male and female offspring in the 100 or 330 mg/kg/day groups.

Mean bodyweights of male and female offspring in the 1000 mg/kg/day group on Days 1 to 13 of age were lower than Control. This is reflected in the overall body weight change; male offspring are 75% of Control and female offspring 74% of Control.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Excluding the clinical signs seen in the pups from the total litter losses, there was a higher incidence of offspring recorded as ‘cold to touch’ in the 1000 mg/kg/day group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Live litter sizes from Day 1 to Day 13 of age were lower than Control for litters from females receiving 1000 mg/kg/day.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
There was a higher incidence of found dead or welfare kills in offspring from the 1000 mg/kg/day group.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
changes in postnatal survival

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for offspring survival, growth and development was 330 mg/kg/day.
Executive summary:

The study was conducted in accordance with the standardized guidelines OECD 422, under GLP conditions to assess the potential systemic toxicity in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by oral 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 in corn 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 or Day 14 of lactation for 3 animals. Females were allowed to litter, rear their offspring and were sacrificed on Day 14/15 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 the treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water 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.

 

Administration of the test item to adult CD:Crl rats at doses ≤ 330 mg/kg/day was generally well tolerated. There were minimal dose observations, no test item-related signs observed during the detailed physical examination and arena observations, and no effects on sensory reactivity, grip strength, motor activity and food consumption.

 

Test item-related findings observed at ≤ 330 mg/kg/day were minimal and low incidence and therefore were not considered adverse. Considering the incidence, severity and significance of the test item-related clinical signs, poor body weight performance, increased water consumption, decreased serum T4 and increased plasma TSH that were evident in the adult animals receiving 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 330 mg/kg/day.

 

Parental test item-related reductions in post implantation survival index, live birth index, viability index, live litter size, offspring bodyweights on Day 1 of age, bodyweight gain and serum T4 were evident in the offspring from females receiving 1000 mg/kg/day. Based on these considerations, the NOAEL for offspring survival, growth and development was 330 mg/kg/day.