Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 2018 - 11 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: AAG0360600
- Expiration date of the lot/batch: 15 March 2018
- Purity : 92.6 %
- Appearance: Clear pale yellow viscous liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cold (~4°C) at ambient humidity in darkness; used/formulated in ambient temperature and light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 180 to 273g
- Fasting period before study: not specified
- Housing: solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 28 January 2018 To: 14 February 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a solution in Propylene glycol. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least twenty days when stored refrigerated at concentrations of 3 mg/mL and 25 mg/mL. However, concentrations of 1.25 mg/mL were found to be stable in ambient conditions for 24 hours. Formulations were therefore prepared at 3.75 mg/mL once, stored at approximately 4 °C and used for the duration of the study. The 3.75 mg/mL solution was diluted daily to produce the concentration of 1.25 mg/mL. The 12.5 mg/mL concentration was prepared twice and stored at approximately 4°C.
Samples were taken of test item formulations on three occasions and were analyzed for concentration of M-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILINE. The results indicate that the prepared formulations were within 96-100 % of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of test item formulations on three occasions and were analyzed for concentration of M-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILINE. The results indicate that the prepared formulations were within 96-100% of the nominal concentration.
Frequency of treatment:
Daily
Duration of test:
Day 5 to Day 19 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages were selected in collaboration with the Sponsor based on available toxicity data including a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat. In the preliminary study, a dosage of 100 mg/kg bw/day was associated with initial body weight loss, lower body weight gains and lower food consumption to Day 14 of gestation, compared to control, sufficient to preclude it from being utilized as a high dosage in this main investigation of pre-natal developmental toxicity. At 50 mg/kg bw/day effects on body weight performance, including a transient body weight loss, and food consumption were apparent at this dosage, but these effects were considered insufficient to exclude this dosage from further investigation of pre-natal developmental toxicity. A previous pre-natal study with a related test item, p-(2,3-epoxypropoxy)-N,N-bis (2,3-epoxypropyl)aniline used dosages of 0 (control), 5, 15 and 40 mg/kg bw/day. As this pre-natal study for M-(2,3-Epoxypropoxy)-N,N-Bis(2,3-Epoxypropyl)Aniline is intended to provide support for read across for this class of test item, the use of similar dosages to those already used was considered to be advantageous, however, given the extent of the responses observed in the preliminary study at 50 mg/kg bw/day, a high dosage of 40 mg/kg bw/day was considered likely to be too low. In view of this a dosage sequence of 0 (control), 5, 15 and 50 mg/kg bw/day was selected, which includes two of the dosages previously investigated with the other test item.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Not specified.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the gestation period, and the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20)


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: The 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
- Other: Placental weight
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.

All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence and distribution of the occasional incidences of clinical signs observed during the study did not show any obvious association with treatment and were considered to be incidental and unrelated to test item exposure.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated at 50 mg/kg bw/day was found dead on Day 17 of gestation. No clinical signs or bodyweight effects had been observed prior to this. At necropsy the lungs were observed to be dark and patchy but the etiology of this death could not be established.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on body weight, body weight gain (both absolute and adjusted for gravid uterus weight) and gravid uterus weight for pregnant females throughout the study at 5, 15 or 50 mg/kg bw/day.
At 50 mg/kg bw/day body weight gain tended to be lower than control from Day 8 of gestation but differences were slight and only attained statistical significance during Days 8 to 11. Although cumulative body weight gain from the start of treatment (Day 5 of gestation) was statistically significantly lower than control on Days 11 and 17 of gestation, overall body weight gain at termination (Day 20 of gestation) was approximately 92 % of control and differences were not statistically significant. In the absence of any significant differences in overall body weight gain when adjusted for the contribution of the gravid uterus, the observed differences in body weight gain were clearly insufficient to represent an adverse effect and may reflect normal biological variation.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on food consumption for pregnant females treated at 5, 15 or 50 mg/kg bw/day.
Females treated at 50 mg/kg bw/day showed slightly lower food consumption, compared to control, during Days 8 to 11 although food consumption for the remainder of the study was generally similar to control. This lower intake, in isolation, was insufficient to represent an adverse effect and may reflect normal biological variation, however, it was consistent with a period of slightly lower body weight gain compared to control.
Food consumption for all treated groups was lower than control between Days 3-5 with values attaining statistical significance at both 5 and 15 mg/kg bw/day, however, the pattern of food consumption observed on the study suggests that the initial food intake for control on the study were higher than would have been expected. Clearly, as this finding was apparent before the commencement of treatment there was no association with treatment and this finding was considered to be incidental and of no toxicological significance.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic necropsy findings for surviving females did not indicate any effect of treatment at dosages of 5, 15 or 50 mg/kg bw/day.
The incidence and distribution of the occasional necropsy findings observed during the study did not show any obvious association with treatment and were considered to be incidental and unrelated to test item exposure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on pre- or post-implantation loss at 5, 15 or 50 mg/kg bw/day.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal weight, placental weight and litter weights were all lower than controls with maternal treatment at 50 mg/kg bw/day, although values did not attain statistical significance.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study the No Observed Adverse Effect Level (NOAEL) for the pregnant female was considered to be 50 mg/kg bw/day. This dosage was also considered to represent a No Observed Effect Level (NOEL) and also the NOAEL for embryofetal survival, growth and development.