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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: grey powder
- Storage conditions of test material: 15 to 25 °C

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:WI(Han)
- Age at study initiation: Females were nine to ten weeks old
- Animal receipt: Females were delivered by Day 3 of gestation
- Weight at study initiation: Females weighed at least 140 g at mating. Females weighed between 167.3 and 252.9 g at the start of dosing.
- Fasting period before study: No
- Housing: The animals were housed singly in cages with Aspen wood chips used for bedding (changed on a weekly basis). Animals were provided with wooden Aspen chew blocks and rodent nesting materials as forms of environmental enrichment.
- Diet: Food was provided ad libitum throughout the study
- Water: Mains water was provided ad libitum via water bottles
- Acclimation period: Acclimatisation was limited by mated status

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 45 to 65 % (relative)
- Air changes: 15 to 20 per hour
- Photoperiod: 12 hrs dark / 12 hrs fluorescent light

IN-LIFE DATES
From: 03 August 2015
To: 21 August 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.1 % CMC (sodium salt)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Frequency of preparation: Formulations were prepared weekly
- Storage of dosing solutions: The formulations were stored refrigerated (2 to 8 °C) in a sealed container
- Test material administration: On arrival at the animal room, the formulations were stirred continuously for thirty minutes before and throughout dosing (excluding the vehicle control group).

VEHICLE
- Amount of vehicle (if gavage): A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
STABILITY AND HOMOGENEITY
- Stability: Determined at 3 and 100 mg/mL for 14 days at 4 °C in the dark in a previous study
- Homogeneity: Determined at 3 and 100 mg/mL in a previous study

CONCENTRATION VERIFICATION
Samples were taken from formulations prepared for the first and last day of dosing. Three samples (1 mL) removed from the test material formulations were added to pre-weighed HPLC vials. These samples were analysed to determine the achieved concentration. Two samples (1 mL) were removed from the control formulations to determine the vehicle subtraction weight.
Details on mating procedure:
- Impregnation procedure: Overnight at the supplier’s laboratory
- Proof of pregnancy: Presence of a vaginal plug in situ, or other evidence of mating, if necessary. The day on which mating was confirmed was designated as Day 0 of gestation.
- Other: Females were considered to be sexually mature at the start of dosing.
Duration of treatment / exposure:
From Day 6 to 20 of gestation
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 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:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level of 1000 mg/kg/day was the No-Observed-Effect-Level (NOEL) in the rat OECD 421 screening study. In the event of treatment related effects, 300 mg/kg/day was considered to be an appropriate step between the low and high dose levels to help characterise findings. The low dose level, 30 mg/kg/day was expected to be a NOEL with respect to adult toxicity and embryo-foetal development.
- Rationale for animal assignment: The animals were assigned to treatment groups on arrival based on body weight and day of mating (i.e. all animals confirmed as mated on a specific day were randomly allocated to treatment groups).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity. The animals were observed at approximately 1 hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on the days of body weight recording. An individual record was maintained of the clinical condition of each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 3, 6, 7, 8, 9, 12, 15, 17, 19, 20 and 21 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The amount of food consumed was determined daily beginning on Day 3 of gestation. Food consumption was calculated as g/animal/day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21. Females were euthanised in replicate order on Day 21 of gestation by cervical dislocation following isoflurane anaesthesia, death confirmed by exsanguination. Food was not removed prior to necropsy. All animals examined macroscopically. All lesions were recorded and retained in the relevant fixative.
- Organs examined: Ovaries and uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and intrauterine position of implantations was subdivided into live foetuses, early intrauterine deaths, late intrauterine deaths and dead foetuses.
Early intrauterine deaths were classified as those which showed decidua or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy. Implantations were allocated numbers relating to their position in utero.
The uterus of any apparently non pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Live foetuses were euthanised by a subcutaneous injection of sodium pentobarbitone.
Individual foetal and placental weights were recorded and foetuses were examined externally and sexed. Approximately one half of the foetuses in each litter, selected by systematic sampling, were dissected and the visceral abnormalities were examined. They were then eviscerated and the carcasses processed for skeletal examination. The skeletons were examined in 50 % glycerol and retained in glycerol/propylene glycol.
The remaining foetuses were fixed in Bouin's fluid and examined. The foetuses fixed for visceral examination were retained in the relevant fixative.
Foetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).
Statistics:
Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data for each sex was analysed separately, unless stated otherwise. Except where otherwise stated, tests were performed using a two-sided risk and were considered significant where P≤0.05.
Body weight, body weight gains and food consumption were analysed using analysis of variance (ANOVA). Mean foetal weight (male, female and combined) was analysed using analysis of covariance (ANCOVA), with litter size (live and dead foetuses) as the covariate. The number of corpora lutea, implantation sites, early and late resorptions, dead foetuses, live foetuses and percent pre- and post-implantation loss and percent male foetuses were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Gravid uterine weights and corrected body weights (carcass weights) were analysed using analysis of variance (ANOVA) and the percentage of foetuses affected (mean litter percent) was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Proportion of litters affected was analysed using a 1–sided upper tail Fishers exact test.
Full details are given below.
Indices:
A number of indices were used, where appropriate, to evaluate reproductive function:
- % pre-implantation loss = [(Number of corpora lutea – number of implantations) / Number of corpora lutea] x 100
- % post-implantation loss = [(Number of implantations – number of live embryos) / Number of implantations] x 100
- Carcass weight = Terminal body weight – uterus weight
- Total weight change = Day 21 body weight – Day 3 body weight
- % male foetuses = (Number of male foetuses / Number of foetuses of determined sex) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations were consistent across groups and unaffected by treatment. Observations consisted primarily of fur and/or coat changes which are common background observations in the rat. There were no post-dosing observations associated with treatment.
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):
Mean body weight parameters were unaffected by the test material at all dose levels.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by the test material at all dose levels.
Food efficiency:
not examined
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
Description (incidence and severity):
There were no macroscopic observations associated with treatment. Caesarean section parameters were consistent across all dose groups and unaffected by treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
DOSE ANALYSIS
The target range for the preparation of the formulations was 90 to 100 % of nominal. All results were within this range and considered acceptable for use.

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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproductive performance was consistent across all groups and unaffected by treatment.
Details on maternal toxic effects:
Sixteen females with implantations per group are considered optimal for statistical analysis of data. In this study there were fifteen females with implantations in the control group. Since the study results are clear, and statistical analysis is not needed to discern the lack of effects on pregnancy and embryo-foetal development, this is not considered to have adversely affected interpretation of the study.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

External malformations:
no effects observed
Description (incidence and severity):
Foetal external parameters were consistent across all groups and unaffected by treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Foetal skeletal parameters were consistent across all groups and unaffected by treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
Foetal visceral parameters were consistent across all groups and unaffected by treatment.

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of Reproductive Performance

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Total Females

20

20

20

20

Pregnant Females

15

17

17

19

Non Pregnant Females

5

3

3

1

Pregnant with Total Litter Loss

1

1

1

0

Females with Live Foeuses

14

16

16

19

Table 2: Summary of Mean Caesarean Section Data

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Number of females pregnant at caesarean section

15

17

17

19

Corpora lutea

12.6

12.9

11.8

12.5

Implantation sites

10.2

11.2

10.9

11.2

Pre-implantation loss

2.4

1.7

0.9

1.3

Pre-implantation loss (%)

18.8

11.5

8.3

11.6

Early resorptions

0.5

0.4

0.6

0.5

Late resorptions

0.0

0.0

0.0

0.0

Total Resorptions

0.5

0.4

0.6

0.5

Dead foetuses

0.0

0.0

0.0

0.0

Post-implantation loss

0.5

0.4

0.6

0.5

Post-implantation loss (%)

11.5

8.7

10.0

4.1

Live foetuses

10.4†

11.5†

10.9†

10.8

†The number of females examined was reduced due to excluded data

 

Table 3: Summary of Mean Foetal Data

Treatment Group

Control

30 mg/kg

300 mg/kg

1000 mg/kg

Number of females with live foetuses

14†

16†

16†

19

Mean number of male foetuses per litter

4.6†

6.2†

5.8†

5.5

Mean number of female foetuses per litter

5.8†

5.3†

5.2†

5.3

% Male foetuses

48.5†

54.1†

53.0†

48.1

Mean foetal weight (g)

5.34†

5.31†

5.41†

5.41

Mean¹ foetal weight - males (g)

5.41†

5.41†

5.51†

5.49

Mean¹ foetal weight - females (g)

5.14†

5.28†

5.28†

5.23

†The number of females examined was reduced due to excluded data

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, daily administration of the test material at doses up to and including 1000 mg/kg/day had no effect on parameters evaluated within this study and the No Observed Adverse Effect Level (NOAEL) is therefore concluded to be 1000 mg/kg/day for both maternal toxicity and developmental toxicity.
Executive summary:

A pre-natal developmental toxicity study was carried out in the rat to investigate the effects of the test material on embryonic and foetal development. The study was conducted in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700 and JMAFF under GLP conditions.

Pregnant female Wistar rats were exposed to the test material in 0.1 % carboxymethylcellulose at dose levels of 0, 30, 300 and 1000 mg/kg/day (20 per group) on Days 6 to 20 of gestation. The females were maintained to Day 21 of gestation when they were sacrificed and their uterine contents examined.

Assessment of toxicity was based on clinical signs, body weight and food consumption. Complete necropsies were performed on all animals with a recording of macroscopic abnormalities for all tissues. Caesarean and foetal examinations were conducted.

There were no unscheduled deaths in the course of the study. Clinical observations, body weight parameters and food consumption were consistent across all groups and unaffected by treatment at doses up to 1000 mg/kg/day.

There were no macroscopic observations associated with treatment and reproductive performance was consistent across all groups and unaffected by treatment. Caesarean section parameters were consistent across all dose groups and unaffected by treatment

Foetal external, visceral and skeletal parameters were consistent across all groups and unaffected by treatment at doses up to 1000 mg/kg/day.

Under the conditions of this study, daily administration of the test material at doses up to and including 1000 mg/kg/day had no effect on parameters evaluated within this study and the No Observed Adverse Effect Level (NOAEL) is therefore concluded to be 1000 mg/kg/day for both maternal toxicity and developmental toxicity.