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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD 414 and GLP compliant study. A default reliability of 2 is assigned for read-across according to ECHA guidance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Substance type: viscous
- Physical state: liquid
- Analytical purity: 100 % UVCB
- Purity test date: 10 Dec 2012
- Batch No.: 240312/K7
- Expiration date of the lot/batch: 19 February 2015
- Stability under test conditions: stable
- Storage condition of test material: stable

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (age at delivery 10 weeks)
- Weight at study initiation: (weight range at delivery 177-196 g)
- Fasting period before study: none
- Housing: individual cages (during gestation)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-05-20 To: 2014-06-16

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.


VEHICLE
- Justification for use and choice of vehicle: The substance is miscible in corn oil and insoluble in water.
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 12.5 to 125 mg/mL by chemical analysis (concentration and
homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits
for concentration (90-110%). Stability after 24 hours at room temperature was verified in the range from 1 to 300 mg/mL in the validation study.
Samples of the formulations prepared on week 1 and Last Week were analysed to check the homogeneity and concentration.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of matingwas made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
Duration of treatment / exposure:
Day 6 through Day 19 post coitum
Frequency of treatment:
daily
Duration of test:
Day 6 through Day 20 post coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding study with pregnant rats. The highest dose group of 500 mg/kg was expected to cause maternal toxicity as indicated by adverse effects on body weights and evidence on liver toxicity as indicated by clinical chemistry parameters. For details it is referred to the robust study summary of the maternal toxicity study.
- Rationale for animal assignment: Females were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION : Yes
- Time schedule for examinations: Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- 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: gross evaluation of placentae, number of intra-uterine deaths,. Uteri or individual uterine horns without visible implantations were immersed
in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the
homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences
between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.
Indices:
Preimplantation loss
Postimplantation loss
Total implantation loss
Sex ratios

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A slight decrease in body weight was noted in all treated females when compared to controls, reaching a statistical significance in females receiving
500 mg/kg bw/day (up to 7%), starting from Day 9 post coitum until the end of the study.
Statistically significant decrease was also recorded in body weight gain of females of the same group on Day 9 post coitum (109%; body weight loss) and Day 12 post coitum (22%). Starting from Day 15 post coitum the mean values of body weight gain were comparable between control and high dose group.

Statistically significant decrease (up to 22%) in food consumption was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum until the end of the study.

A slight trend to decrease was observed in terminal body weight of all treated females with respect to the control. This change was about -6% in the high
dose group, without statistical significance. A statistically significant decrease in corrected body weight (up to 6%) and corrected body weight gain (up
to 50%) was noted in treated females receiving 150 and 500 mg/kg bw/day. Gravid uterus weight was similar between control and treated groups.

There were no adverse findings at the macroscopic examination at necropsy.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter data, mean foetal weight and sex ratio were unaffected by treatment.

A total of 15 small foetuses (foetal weight < 2.7 g) were detected: 2 out of 269 in the control group, 2 out of 214 in the low dose group, 1 out of 269 in the mid-dose group and 10 out of 254 in the high dose group. One foetus in the high dose group showed malrotation of the hindlimb, considered incidental. Of the ten small fetuses in the high dose group, 7 were from the dam which suffered most strongly from maternal toxicity as indicated by the lowest corrected body weight gain of minus 9.8g. This dam was also the only dam showing hunched posture and piloerection on gestation day 20. The higher incidence of small foetuses is therefore considered to be related to the lower maternal body weight gain.

No relevant findings that could be considered treatment-related were observed at visceral examination of foetuses in the treated groups, compared to controls.
The alterations recorded at skeletal examinations of foetuses were noted both in control and treated groups with a similar incidence.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Remarks on result:
other: No teratogenicity at the hightest tested dose
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryotoxicity
Remarks on result:
other: No embrotoxicity at the highest tested dose

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

Group   Terminal body weight (g) Gravid uterus weight (g) Body weight at necropsy minus gravid uterus weight (corrected body weight) (g)  Body weight at necropsy minus gravid uterus weight, minus body weight on GD6 (corrected body weight gain) (g) 
1 (control) Mean 336.38 66.40 269.98 29.87
  SD 15.84 8.80 14.79 7.67
  (n) 23 23 23 23
2 Me 323.9 60.42 263.5 29.45
  SD 23 18 18 16
  (n) 20 20 20 20
3 Me 323.8 66.20 257.6* 20.5*
  SD 28 12 18 9.
  (n) 23 23 23 23
4 Me 315.9 62.53 253.3* 14.83*
  SD 24 14 23 16
  (n) 22 22 22  22

* = Statistically significantly different from control group value at p< 0.05

Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN

Group   gestation day 3 gestation day 6 gestation day 9 gestation day 12 gestation day 15 gestation day 18 gestation day 20
1 (control) n  23 23 23 23 23 23 23
  Mean 18.13 20.66 18.72 20.99 21.78 23.68 23.06
  SD 2.37 2.54 2.05 1.98 2.10 2.20 1.99
2 n  20 20 20 20 20 20 20
  Mean 17.99 20.97 18.32 20.64 21.04 23.43 22.83
  SD 2.3 3.03 2.46 2.19 2.68 2.99 4.33
3 n  23 23 23 23 23 23 23
  Mean 18.7 20.23 17.36 19.28 20.90 22.32 21.56
  SD 1.99 2.53 2.59 2.90 2.40 2.91 3.14
4 n  22 22.00 22 22.00 22.00 22.00 22.00
  Mean 18.24 20.97 15.33** 17.1** 19.01** 20.12** 18.02**
  SD 2.62 2.55 1.98 2.76 2.68 3.02 2.96

** = mean value of group is significantly different from control at p < 0.01

Table 3: Pregnancy status overview

Group 1 (control) 2 3 4
Initial group size (n) 24 24 24 24
Not pregnant (n) 1 4 1 2
Unilateral implantation (n) 0 1 0 0
With live foetuses at gestation Day 20 (n) 23 20 23 22

Table 4: Litter data and sex ratios - group mean data

    Corpus lutea Implantations Early Uterine Deaths Late Uterine Deaths Total Uterine Deaths Viable Young (total) Viable males Viable females % Males Preimplantation loss (%) Postimplantation loss Total implantation loss(%) Litter weight (g) Mean fetal weight (g)
1 Mean 12.87 12.26 0.52 0.04 0.57 11.7 6 5.7 50.96 4.68 4.15 8.52 43.1 3.69
  SD 1.63 1.63 1.47 0.21 1.5 1.77 2.24 2.05 17.27 5.13 10.27 11.78 6.71 0.34
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
2 Mean 12.26 11.47 0.21 0.05 0.26 11.21 5.67 5.84 49.82 6.56 4.12 10.05 39.88 3.63
  SD 2.58 2.44 0.54 0.23 0.56 2.84 2.22 2.29 14.18 6.13 11.67 13.68 9.32 0.4
  (n) 19 19 19 19 19 19 18 19 18 19 19 19 19 19
3 Mean 13.09 12.22 0.52 0 0.52 11.7 5.78 5.91 449.19 6.28 4.73 10.86 42.41 3.65
  SD 2.7 2.35 0.73 0 0.73 2.55 1.93 1.98 12.79 6.68 6.74 7.39 8.85 0.28
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
4 Mean 12.27 11.73 0.14 0 0.14 11.59 5.36 6.23 46.95 6.11 1.18 7.23 40.11 3.51
  SD 2.66 3.06 0.35 0 0.35 3.08 2.06 2.25 12.97 10.6 3.07 10.96 9.86 0.39
  (n) 22 22 22 22 22 22 22 22 22 22 22 22 22 22

Applicant's summary and conclusion

Conclusions:
The substance is not teratogenic and not embryotoxic in rats. It causes maternal toxicity at a dose level of 500 mg/kg bw.