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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:
28 Feb - 02 Jul 2014
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Deviations:
yes
Remarks:
six dams were dosed at a lower dose level than intended on first day of dosing (63% of nominal concentration)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1998)
Deviations:
yes
Remarks:
six dams were dosed at a lower dose level than intended on first day of dosing (63% of nominal concentration)
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (2000)
Deviations:
yes
Remarks:
six dams were dosed at a lower dose level than intended on first day of dosing (63% of nominal concentration)
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Limit test:
no

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Y-15099
- Physical state: pale yellow liquid
- Analytical purity: 95.4%
- Lot/batch No.: 13NSUN161
- Expiration date of the lot/batch: 18 Dec 2017
- Storage condition of test material: at room temperature in the original container protected from light under nitrogen headspace

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD® (SD) IGS BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Weight at study initiation: 228 - 308 g
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum

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

IN-LIFE DATES: From: 23 Mar 2014 To: 10 Apr 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: desiccated Arachis oil BP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in dessicated Arachis oil BP. Test material formulations were proven to be stable for at least twenty days. Formulations were therefore prepared within the stability period and stored at approximately 4 °C in the dark under nitrogen headspace.

VEHICLE
- Concentration in vehicle: 6, 30 and 150 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test material gave a chromatographic profile consisting of a single peak. The formulations investigated during the study were found to comprise test material in the range of 96 to 104%, and thus, the required content limit of ±10% with reference to the nominal content was met. An exception was recorded on the first day of dosing when six female animals of the low dose group were exposed to 63% of the nominal concentration only. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
Animals were delivered in two batches containing females prior to Day 2 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
Day 5-19 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
until Day 20 of gestation
No. of animals per sex per dose:
24 females/group with
24 pregnant females in the control group
21 pregnant females in the low dose group
22 pregnant females in the mid dose group
23 pregnant females in the high dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels and treatment volume were chosen based on the available data from a 28 Day study (RCC study Number: 808378, November 2001).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on 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).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 of gestation
- Organs examined: All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed and examined. The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
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
- Fetal sex: Yes
- External fetal appearance: Yes
- Fetal weight: Yes
- Placental weight: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
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, an alternative multiple comparison test. 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<0.01 = **, p<0.05 = *, p=0.05 (not significant).
Indices:
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 fetuses) / number of implantations] x 100
Sex ratio (% male fetuses) = (number of male fetuses / total number of fetuses) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: (non-adverse)

Details on maternal toxic effects:
There were no treatment-related deaths during the study period. One female of the low dose was killed on Day 6 of gestation due to a decline in clinical condition. Clinical signs prior to death included hunched posture, piloerection, laboured and noisy respiration, decreased respiratory rate and staining around the snout. At necropsy, macroscopic findings were consistent with physical trauma following dosing procedure and thus, the death of this animal was considered unrelated to treatment with the test material.
There were no clinical signs considered to be related to the toxicity of the test material at any dose level. Increased post-dose salivation was seen in two females of the high dose group on Days 6 and 7 of gestation (one animal on each day). No such effects were detected in females of the low and medium dose groups. Observations of this nature are commonly observed following the oral administration of an unpalatable test material formulation, and in isolation, are considered of no toxicological importance. One female of the medium dose group showed signs of chromodaccryorrhea (Day 13 of gestation) and swollen snout and right side of the face (Days 13 to 16 of gestation); these observations were considered to be associated with a physical injury attained during the course of the study and hence are considered unrelated to treatment with the test material. A female of the low dose group showed signs of noisy respiration between Days 6 to 9 of gestation. In isolation, this was considered not to be related to treatment with the test material. No toxicologically significant effects of treatment with the test material on body weight development were detected at any dose level. Females of the high dose group showed slight group mean body weight loss over the first day of dosing, but without achieving statistical significance when compared with controls. Subsequent improvement in body weight was apparent for these females such that the mean gains from Day 8 of gestation were comparable with controls. Group mean cumulative body weight gains for these females were statistically significantly lower than controls over Days 5 to 7 (p<0.01), 5 to 8 (p<0.01) and 5 to 11 (p<0.05) of gestation. Since the initial effect on body weight development was minimal with full recovery evident thereafter, this finding was considered not to be of any toxicological significance. There was no effect of treatment with the test material on body weight development in females given the test material at 150 or 30 mg/kg bw/day. There was no detrimental effect of treatment with the test material food intake at any dose level. Over Days 5 to 8 of gestation, group mean food consumption in females of the low and medium dose groups was slightly but statistically significantly higher than controls (p<0.05 and p<0.01, respectively). In contrast, the mean food intake in females of the high dose group over the corresponding period was similar to controls. Over the remaining days, food intake across all treated groups was similar to controls. Daily visual inspection of water bottles did not reveal any overt intergroup differences. There were no macroscopic findings for any of the animals surviving to the scheduled necropsy on Day 20 of gestation with exception of one female (see above).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: (non-adverse)

Details on embryotoxic / teratogenic effects:
In total, out of a total number of 24 females per dose group, 24, 21, 22 and 23 females from the control, low, medium and high dose groups, respectively, were found to be pregnant at scheduled necropsy on Day 20 of gestation. One female of the low dose group that was killed in extremis on Day 6 of gestation was also pregnant. The incidence and distribution of non-pregnant females was considered to be within the background ranges for this strain of animals. There were no treatment-related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. Pre-implantation losses and sex ratios across all treated groups were similar to controls. Fetal or placental weights were also unaffected by treatment with the test item. For all dose groups, there were no statistically significant treatment-related trends in the proportion of fetuses (or litters) with evidence of external, visceral or skeletal anomalies. At the high dose group, major malformations were confined to one fetus showing microphthalmia. This abnormality is known to occur spontaneously in this strain of rat, and due to its isolated nature in this study, it was considered unlikely to be related to treatment with the test item. At the low dose group, one fetus was noted with an absent kidney and ureter. Since this finding was not present in any of the fetuses from the medium or high dose groups, it was considered unrelated to treatment with the test item. Other external, visceral and skeletal anomalies were either similar to controls and/or commonly observed for this type of study.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: No effects noted, highest test group

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

Table 1: Summary of female performance

Category

Number of females at dose level (mg/kg bw/day)

0 (Control)

30

150

750

Initial Group

Size

24

24

24

24

Pregnant

24

22

22

23

Number with

Fetuses on Day 20 of Gesation

24

21

22

23

Table 2: Group mean litter data values

Dose level (mg/kg bw/day)

 

Number of Corpora Lutea

Number of Implants

Number of Embryonic/Fetal Deaths

Implantation Loss (%)

Number of Live Implants

Male Fetuses (%)

Mean Male Fetal Weight (g)

Mean Female Fetal Weight (g)

Mean Fetal Weight (g)

 

Mean Placental Weight

(g)

Litter Weight (g)

Total Placental Weight

(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0

Mean

SD

N

14.0

4.0

24

13.4

4.0

24

0.2

0.6

24

0.0

0.0

24

0.2

0.6

24

5.7

10.0

24

1.3

3.6

24

6.7

2.5

24

6.5

2.7

24

13.2

3.9

24

52.8

15.5

24

4.222

0.256

24

3.936

0.248

23

4.092

0.262

24

0.589

0.178

24

53.575

15.381

24

7.173

1.911

24

30

Mean

SD

N

15.4

1.9

21

14.4

1.6

21

0.2

0.5

21

0.1

0.4

21

0.3

0.6

21

6.2

4.9

21

2.6

4.6

21

6.3

2.2

21

7.7

2.0

21

14.0

1.6

21

44.8

13.3

21

4.200

0.242

21

3.999

0.215

21

4.091

0.227

21

0.557

0.049

21

57.445

7.256

21

7.814

1.086

21

150

Mean

SD

N

15.1

1.3

22

14.3

1.8

22

0.2

0.5

22

0.1

0.4

22

0.3

0.7

22

5.6

5.7

22

2.0

4.3

22

7.6

2.4

22

6.4

1.8

22

14.0

1.5

22

53.8

14.7

22

4.204

0.333

22

3.980

0.321

22

4.095

0.308

22

0.549

0.050

22

57.090

6.798

22

7.630

0.811

22

750

Mean

SD

N

14.4

3.1

23

13.5

3.5

23

0.1

0.3

23

0.1

0.3

23

0.4

0.7

23

8.6

14.4

23

2.8

4.6

23

6.9

2.1

23

6.3

2.2

23

13.1

3.4

23

54.4

15.0

23

4.210

0.399

23

3.923

0.171

22

4.108

0.410

23

0.562

0.083

23

52.762

12.663

23

7.158

1.730

23

SD: Standard deviation

N: number of pregnant animals

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of the test item to pregnant female Sprague-Dawley rats, at dose levels of 30, 150 or 750 mg/kg bw/day was well tolerated. No toxicological relevant effects were recorded, thus a dose level of 750 mg/kg bw/day is considered to be the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity.
Executive summary:

The test item was administered by gavage to three groups of 21, 22 and 23 pregnant female Sprague-Dawley Crl:CD® (SD) IGS BR rats, during gestation days 5-19, at dose levels of 30, 150 or 750 mg/kg bw/day, respectively. A control group of 24 pregnant female rats was dosed with vehicle alone (dessicated Arachis oil BP). Clinical signs, body weight development, dietary intake and water consumption were monitored during the study period. On day 20 of gestation the animals were euthanized and examined for maternal and fetal parameters. There were no adverse effects found for all parameters examined in maternal animals. Based on the number of implantations, number of total litter losses by resorption, mortality, gravid uterus weight, number of corpora lutea, placenta weight clinical signs, body weight, and gross pathology of maternal animals the NOAEL for maternal toxicity was found to be 750 mg/kg bw/day. Examination of fetus litter size and weights, offspring viability (number alive and number dead), sex ratio, grossly visible abnormalities, external, head, soft tissue and skeletal abnormalities showed only incidental malformations in each one low and high dose fetuses. Thus, the NOAEL for developmental toxicity in rats for the test item was found to be 750 mg/kg bw/day.