Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
The developmental toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 March 2015 to 07 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: JMAFF, 12 - NohSan No. 8147 (2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 180 g to 246 g
- Fasting period before study: No
- Housing: Females were housed individually in grid-floor cages over paper lined trays.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 40% to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % aqueous carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at approximately weekly intervals (within the known stability period), for each group separately, as a suspension in 1 % aqueous carboxymethylcellulose.
A weighed quantity of test item was added to a mortar, wetted with a small quantity of vehicle and initially made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a tared beaker on a balance.
The mortar was thoroughly rinsed out with vehicle and these rinsings were added to the suspension which was then made up to final weight with vehicle and mixed with a laboratory homogeniser.
Formulations were divided into daily aliquots and were stored frozen (approximately -20 °C) or, if to be used the following day, formulations were stored at room temperature, protected from light. Frozen aliquots were removed from storage on the afternoon before dosing and stored at room temperature overnight to defrost. Formulations were stirred from 30 minutes before the start of dosing, until the completion of their use for dosing, to ensure thorough resuspension and homogeneity.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first preparation occasion, test item formulations were analysed to assess homogeneity and achieved concentration of test item, using a validated method (BFI009LC (4)). Once homogeneity had been established, subsequent formulations for use on the last day of dosing were analysed to determine achieved concentrations only. Vehicle (for Controls) from the first preparation and that for use on the last day of dosing was analysed to confirm the absence of the test item.
All remaining samples were retained frozen (approximately -18 ºC) and discarded once the final formulation analysis results were accepted.
Details on mating procedure:
Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.
Duration of treatment / exposure:
Day 6 of gestation to Day 19 of gestation
Frequency of treatment:
once daily
Duration of test:
From Day 0 of gestation to Day 20 of gestation (necropsy)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in consultation with the Sponsor on the basis of results from a reproduction / developmental toxicity screening test and a two-generation reproduction toxicity study in the rat performed in the same laboratory.
- Rationale for animal assignment (if not random): Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on Day 0 of gestation, making sure that females mated with the same male were spread across the
groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity and were given a detailed clinical examination daily from the start of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 0 of gestation by the supplier. At Sequani, body weights were recorded daily from Day 5 to 20 of gestation, inclusive.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food intake was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, and 18 to 20 of gestation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The major organs were examined. Gravid uterus weight and placenta weights were recorded and organs or tissues showing macroscopic abnormalities were removed and retained in neutral buffered formaldehyde. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to
confirm pregnancy status. The uterus was retained in 70 % IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in neutral buffered formaldehyde.

Ovaries and uterine content:
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses.
The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
Fetal examinations:
- External examinations: Yes: All
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Approximately 50 % of the live foetuses were fixed in Bouin’s fluid for subsequent examination for visceral abnormalities using a combined sectioning/dissection technique.
The remaining foetuses were briefly fixed in 70 % alcohol and subjected to micro-dissection. The viscera were examined and the foetuses were eviscerated. The carcasses were then cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined for skeletal variants and abnormalities.
Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Other defects were classified as minor abnormalities or variations.
- Head examinations: No data
Statistics:
All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. The litter, rather than the foetus, was considered as the experimental unit. Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA).

Dunnett’s test: For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett’s test was used to compare the control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett’s test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant, and statistical flags are presented in the tables of results in the final report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female given 1000 mg/kg/day (Animal 78) had piloerection on Day 8 of gestation; as this female also had marked initial body weight loss, an association with treatment would seem likely, however, since this was an isolated occasion it was considered not to be adverse. There were no other clinical signs during the study at any dose level.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Over Days 6 to 8 of gestation, there was a slight mean body weight loss in the group given 1000 mg/kg/day, with notable intra animal variation. From Day 9 of gestation onwards, group mean body weight gain was comparable with Controls; therefore, the initial body weight effect at 1000 mg/kg/day was considered not to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the group given 1000 mg/kg/day, animals ate statistically significantly less over Days 6 to 9 of gestation (p<0.01); thereafter, food intake was similar to Controls.
At 50 or 250 mg/kg/day, there was no effect of CA4920 on food intake.
Food efficiency:
no effects observed
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Two females (Control Female 14 and Female 74 given 1000 mg/kg/day) had total early intrauterine deaths and two females were not pregnant (Female 63 given 250 mg/kg/day and Female 73 given 1000 mg/kg/day). Consequently, there were 21, 22, 21 and 20 females in the groups given 0, 50, 250 or 1000 mg/kg/day respectively, with live foetuses on Day 20 of gestation. There were no effects of the test item on pregnancy parameters, the numbers of implantations, incidence of pre- and post-implantation loss and numbers of live foetuses were similar in all groups.
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)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs

Maternal abnormalities

Abnormalities:
no effects observed

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:
effects observed, non-treatment-related
Description (incidence and severity):
Only one major foetal abnormality was present in one foetus given 250 mg/kg/day (the hind limb fibula(e) was not ossified) and there were no statistically significant differences from Control in the overall incidences of minor or variant foetal abnormalities in the treated groups.
In the groups given 250 or 1000 mg/kg/day, there was a significant (p<0.05) increase in the number of litters with foetuses showing the variant finding of increased renal pelvic cavitation. At 1000 mg/kg/day, there was also a significant increase (p<0.05) in the numbers of litters with foetuses having an extra or vestigial 14th rib (with costal cartilage), one or more of the 11th to 13th ribs with long costal cartilage and incomplete cartilaginous styloid process(es) in the skull. The number of foetuses showing these minor or variant abnormalities was either very small, lacked any dose response or were within or just marginally above the background data ranges.
These findings do not indicate an effect of the test item on foetal development.
Visceral malformations:
no effects observed

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull
Description (incidence and severity):
Only one major foetal abnormality was present in one foetus given 250 mg/kg/day (the hind limb fibula(e) was not ossified) and there were no statistically significant differences from Control in the overall incidences of minor or variant foetal abnormalities in the treated groups.
In the groups given 250 or 1000 mg/kg/day, there was a significant (p<0.05) increase in the number of litters with foetuses showing the variant finding of increased renal pelvic cavitation. At 1000 mg/kg/day, there was also a significant increase (p<0.05) in the numbers of litters with foetuses having an extra or vestigial 14th rib (with costal cartilage), one or more of the 11th to 13th ribs with long costal cartilage and incomplete cartilaginous styloid process(es) in the skull. The number of foetuses showing these minor or variant abnormalities was either very small, lacked any dose response or were within or just marginally above the background data ranges.

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Maternal changes seen at 1000 mg/kg/day were considered to be transient and of no adverse outcome.There was no adverse effect on pregnancy or embryonic or foetal development. On this basis, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and No Observed Effect Level (NOEL) for embryo-foetal development was considered to be 1000 mg/kg/day.
Executive summary:

Four groups of 22 sexually mature timed-mated female Crl:WI(Han) rats were dosed with 0 (vehicle), 50, 250 or 1000 mg/kg/day of the test item, once daily by oral gavage, from Days 6 to 19 of gestation, inclusive.

All animals were observed daily from the start of treatment and body weights and food intake were recorded throughout. Animals were killed on Day 20 of gestation, a necropsy performed and the internal organs examined for gross abnormalities. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral and skeletal abnormalities. Placental and gravid uterus weights were also recorded.

Slight mean body weight loss was evident after the first two days of dosing at 1000 mg/kg/day and less food was eaten over this period, from Day 9 of gestation onwards, body weight gain and food intake were similar to controls.

There were 21, 22, 21 and 20 females in the groups given 0, 50, 250 or 1000 mg/kg/day, respectively, with live foetuses on Day 20 of gestation. There were no effects on pregnancy parameters; values in all groups were similar. At all dose levels, mean foetal and placental weights were comparable with those of the Control group and there was no effect on foetal sex ratio.

There were no maternal macroscopic abnormalities related to the test item. There was no adverse effect of the test item on the incidence of external, visceral, skeletal or cartilaginous major or minor foetal abnormalities or variants.