Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 July 2016 and 25 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
FAT Number: 92504/C TE
Appearance: blue powder
The test item was measured with a 0,25 % (w/w) solution
Specific details on test material used for the study:
Identification : FAT 92504/C TE
Physical State/Appearance: Blue paste
Purity: 86.4%
Batch Number: BOP 01-15
Label: FAT-Nr. 92504/C TE Lot. Nr.: BOP 01-15 bei
Raumtemperatur VD: 18.11.2020 (Retest Date)
Date Received: 14 March 2016
Storage Conditions: Room temperature, in the dark (14 March 2016 – 18 March 2016). Stored cold at
approximately 4°C in the
dark (18 March 2016 onwards)
Expiry Date: 18 November 2020
No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on species / strain selection:
As recommended by the guideline
Sex:
male/female
Details on test animals and environmental conditions:
The animals were acclimatized for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 310 to 348 g, the females weighed 186 to 226g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided
in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding,
throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour
and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and
print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol
400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL)
Animal Numbers
Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 1000 4 250 12 (73-84) 12 (85-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group. The test item was administered daily by gavage using a stainless steel cannula attached to a disposable
plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least nineteen days.
Formulations were initially prepared for one week and then fortnightly thereafter and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken and analyzed for concentration of FAT 92504/C TE at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.
Duration of treatment / exposure:
Up to eight weeks
Frequency of treatment:
Daily (except for females during parturition where applicable)
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was
designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Days 43 and 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Examinations

Parental animals: Observations and examinations:
None
Statistics:
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs for any of the animals considered to be related to the toxicity of the test item.
For all treatment groups, instances of blue fur staining by the test item were frequently observed in both
males and females from the first week of dosing at 1000 and 300 mg/kg bw/day and from the second
week of dosing at 100 mg/kg bw/day. Blue colored faeces and staining of the cage bedding were also
observed for animals of either sex from all treatment groups from the first week and second week of
dosing (respectively). These observations generally persisted throughout the remainder of the study.
One female treated with 100 mg/kg bw/day had increased salivation post dosing on Day 45 and one male
from this treatment group had pilo-erection between Days 29 and 37. In isolation and in the absence of
a similar effect at 300 or 1000 mg/kg bw/day, these observations were considered to be incidental and
unrelated to treatment. One control male, two males treated with 100 mg/kg bw/day and one male treate
d with 300 mg/kg bw/day showed incidences of noisy respiration. In the absence of a similar effect in
females or in either sex at 1000 mg/kg bw/day this finding was deemed likely to be due to the dosing
procedure.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on body weight development in
animals of either sex.
Males from all treatment groups showed a statistically significant increase in body weight gain (p<0.05)
during Week 4 of treatment. An isolated incidence of increased body weight gain is considered not to
represent an adverse effect of treatment
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption or food conversion efficiency (where calculated) in animals of either sex.
Statistical analysis of the data (where applicable) did not indicate any significant intergroup differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any intergroup differences in water intake for animals of either sex given the test item in relation to controls.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the hematological parameters examined. Statistical analysis of the data did not reveal any significant intergroup differences.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in chloride
concentration (p<0.05). Males treated with 1000 mg/kg bw/day also showed a statistically significant reduction in calcium concentration (p<0.05). All of the individual values were within background control
ranges and in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment at any dose level.
Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
During motor activity evaluations, males treated with 1000 mg/kg bw/day showed a statistically significant increase during the final 20% of activity monitoring time (p<0.05) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup difference was considered not to be of toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic abnormalities detected.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the
integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the
ovaries.
Other effects:
effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

Mating
There was no effect of treatment on mating performance with all animals mating within four days after pairing.
Fertility
No treatment-related effects were detected in fertility.
One female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day were not
pregnant following positive evidence of mating. There was evidence of uterine dilation and inflammation
in the female treated with 100 mg/kg bw/day which may indicate a problem following mating and is most
likely to be the reason for the lack of pregnancy. No histopathological changes were evident in the female treated with 300 mg/kg bw/day however the male partner had tubular atrophy in the testes which was
the reason for the lack of pregnancy. In the absence of any fertility effects at 1000 mg/kg bw/day, these
intergroup differences were considered to be incidental.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
Statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.

Litter Responses
In total twelve females from the control group, eleven females each from the 100 and 300 mg/kg bw/day
dose groups and twelve females from the 1000 mg/kg bw/day dose group gave birth to a live litter and
successfully reared young to Day 5 of age. Female 22 was administered test item on Day 3 of lactation
due to a technical error. The data for this female has therefore been excluded from assessment from
Day 3 of lactation onwards. The following assessment of litter response is based on all remaining litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation
losses, litter size or litter viability for treated animals when compared to controls. Statistical analysis of
the data did not reveal any significant intergroup differences.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated
groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Offspring Growth and Development
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to
controls. The incidental clinical signs detected throughout the control and treated groups, consisting
of small size, pale, physical injury, missing or found dead were considered to be low incidence findings
observed in offspring in studies of this type and were considered unrelated to test item toxicity. On Day
5 of lactation, four litters from females treated with 1000 mg/kg bw/day were stained blue. This was
considered to be the result of the administration of a colored test item to the adult female and the transfer
of the colored material to the offspring following the dams normal grooming procedure.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
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)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Days 43 and 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

The oral administration of FAT 92504/C TE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day for either sex. The NOEL (No Observed Effect Level) for reproductive toxicity was determined to be 1000 mg/kg bw/day.