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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27th October 2017 - 14 February 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 12 NouSan No 8147
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2-methylfuran
EC Number:
202-507-4
EC Name:
Tetrahydro-2-methylfuran
Cas Number:
96-47-9
Molecular formula:
C5H10O
IUPAC Name:
2-methyltetrahydrofuran
Details on test material:
- Name of test material (as cited in study report): 2-methyltetrahydrofuran; MeTHF CAS 96-47-9
- Substance type: Monoconstituent substance
- Physical state: no information
- Analytical purity: no information
Specific details on test material used for the study:
Identification : MeTHF
Appearance/Physical State : Clear colorless/liquid
CAS: 90-47-9
Chemical Name: Tetrahydro-2-methylfuran
Alternative Identification : 2-Methyltetrahydrofuran
Purity: 99.98%
Batch : 2-7E25S
Date Received : 07 June 2017
Storage Conditions : Ambient temperature and humidity in darkness; used/formulated in light
Expiry Date : 25 May 2019

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Time-mated females
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Not reported
- Weight at study initiation: 187-294 g
- Fasting period before study: None
- Housing: Single housed in solid-floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet: ad libitum Rodent 2018C Teklad Global diet
- Water: ad libitum mains drinking water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 03-23 November 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test material was formulated in arachis oil, based on solubility, at a constant volume of 4mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo. Formulations were shown to be stable for at least eleven days when stored refrigerated. Formulations were therefore prepared in batches up to every eight days, divided into daily aliquots, and stored at approximately 4°C in the dark. Samples were taken of each test item formulation on two occasions and were analysed for test material concentration. The results of the analyses indicate that the prepared formulations were within 96-99% of the nominal concentration.
Details on mating procedure:
Time-mated female Sprague-Dawley Crl:CD(R)(SD) IGS BR strain rats were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation (GD0).

Duration of treatment / exposure:
The test material was administered daily, from Day 3 (prior to implantation) to Day 19 of gestation, by gavage.
Frequency of treatment:
Daily
Duration of test:
All animals were terminated on GD20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle (arachis oil) control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
24 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Rats were randomly allocated to treatment groups using a randomidation procedure based on stratified body weight to ensure similarity between the treatment groups. Dose levels were based on the resuts of a 14-day oral range-finding study and a preliminary study in pregnant rats.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Following arrival, 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. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
Food consumption was recorded for each individual animal on Days 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION: Yes
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes

All implantations and viable fetuses were numbered according to their intrauterine position
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
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: the 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. 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 nonparametric analysis of variance and Mann-Whitney ‘U’ test. Probability values (p) were presented as follows: p<0.01 **; p<0.05 *; p=0.05 (not significant)
Indices:
Not applicable
Historical control data:
Historical control data (updated 2014) for foetal findings are provided in the study report. Specific reference is made to the historical range for nasal ossification (1605 foetuses from 255 litters).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 1000 mg/kg bw/d. At 1000 mg/kg bw/d, seven animals were noted to exhibit a ploughing motion with the snout immediately post-dosing and five animals showed increased salivation post-dosing. These signs were considered to reflect either a palatability effect or slight irritancy of the test material and not to be adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, body weight gain was statistically significantly lower than controls on the first day of treatment, but recovery of body weight gain was apparent the following day. Body weight gains were again lower than control from Day 14 to termination, although only values for Days 14-17 attained statistical significance, and resulted in statistically significant lower cumulative body weight gain from Day 17 of gestation. To a certain extent, these differences in body weight gain during late gestation may reflect a lower contribution from the litter, as gravid uterus weight was statistically significantly lower than control at this dose level. When adjusted for the contribution of the gravid uterus, although final body weight and overall body weight gain from the start of treatment remained lower than controls, differences no longer attained statistical significance.

At 300 mg/kg bw/d, body weight gain was statistically significantly lower than controls on the first day of treatment, but recovery of body weight gain was apparent the following day. There was no subsequent effect on weight gain and overall body weight gain, including when adjusted for the contribution of the gravid uterus, was unaffected by treatment.

At 100 mg/kg bw/d, there was no effect on body weight gain during gestation, including overall body weight gain when adjusted for the contribution of the gravid uterus.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect on food consumption during gestation at 100, 300 or 1000 mg/kg bw/d.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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):
Macroscopic necropsy findings observed for adult animals did not indicate any effect of treatment at 100, 300 or 1000 mg/kg bw/d.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
There were no unscheduled deaths on the study. Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 1000 mg/kg bw/d. At 1000 mg/kg bw/d, seven animals were noted to exhibit a ploughing motion with the snout immediately post-dosing and five animals showed increased salivation post-dosing. These signs were considered to reflect either a palatability effect or slight irritancy of the test material and not to be adverse.At 1000 mg/kg bw/d, body weight gain was statistically significantly lower than controls on the first day of treatment, but recovery of body weight gain was apparent the following day. Body weight gains were again lower than control from Day 14 to termination, although only values for Days 14-17 attained statistical significance, and resulted in statistically significant lower cumulative body weight gain from Day 17 of gestation. To a certain extent, these differences in body weight gain during late gestation may reflect a lower contribution from the litter, as gravid uterus weight was statistically significantly lower than control at this dose level. When adjusted for the contribution of the gravid uterus, although final body weight and overall body weight gain from the start of treatment remained lower than controls, differences no longer attained statistical significance. At 300 mg/kg bw/d, body weight gain was statistically significantly lower than controls on the first day of treatment, but recovery of body weight gain was apparent the following day. There was no subsequent effect on weight gain and overall body weight gain, including when adjusted for the contribution of the gravid uterus, was unaffected by treatment. At 100 mg/kg bw/d, there was no effect on body weight gain during gestation, including overall body weight gain when adjusted for the contribution of the gravid uterus. There was no effect on food or water consumption in any group. Macroscopic necropsy findings observed for adult animals did not indicate any effect of treatment at 100, 300 or 1000 mg/kg bw/d.

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:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, fetal weights were slightly lower than control with differences for female and combined weights attaining statistical significance. This resulted in a lower gravid uterus weight and litter weight compared to control, although only differences for the gravid uterus attained statistical significance. Total placental weight was also statistically significantly lower than control at this dosage but, this occurred in the absence of any significant effect on mean placental weight.
There was no effect of maternal treatment on mean fetal, placental and litter weight on Day 20 of gestation at 100 or 300 mg/kg bw/d.
Details on maternal toxic effects:
There was no effect of maternal treatment on litter data, as assessed by pre-implantation loss, number of implantations, subsequent embryofetal survival, live litter size and sex ratio on Day 20 of gestation at 100, 300 or 1000 mg/kg bw/d.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
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)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, fetal weights were slightly lower than control with differences for female and combined weights attaining statistical significance.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d, litter weights was slightly lower than control with the difference attaining statistical significance.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The type, incidence and distribution of external findings at 100, 300 and 1000 mg/kg bw/d did not indicate any obvious effect on fetal development.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The type, incidence and distribution of skeletal findings at 100, 300 and 1000 mg/kg bw/d did not indicate any obvious effect on fetal development.
Visceral malformations:
no effects observed
Description (incidence and severity):
The type, incidence and distribution of visceral findings at 100, 300 and 1000 mg/kg bw/d did not indicate any obvious effect on fetal development
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
At 1000 mg/kg bw/d, the incidence of fetuses showing incomplete nasal ossification (15.1%) was statistically significantly higher than control. However, although the incidence of this finding for litters at 1000 mg/kg bw/d exceeded the historical control range (0-6.3%; mean 2.6%), so did the incidences for the control (8.3%) and 300 mg/kg bw/d litters (8.2%). While there was some variation from control for some other ossification parameters at 1000 mg/kg bw/d, there was no obvious effect on the overall pattern of ossification and, therefore, this isolated finding, although supported by slightly lower fetal weight, appears most likely to be incidental and unrelated to maternal treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
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

Summary of findings

Dose level (mg/kg bw/d)

0

100

300

1000

Mated (#)

24

24

24

24

Not pregnant (#)

0

1

0

1

Litters (#)

24

23

24

23

Salivation (#)

0

0

0

5

Ploughing (#)

0

0

0

7

Bodyweight (g)

GD3

247.1

248.1

250.4

246.3

GD4

254.1

253.2

255.0

249.9

GD5

259.1

259.8

260.5

255.0

GD8

270.2

272.9

272.4

266.0

GD20

378.7

386.0

379.4

363.3

Bodyweight gain (g)

GD3-4

7.0

5.0

4.6*

3.6**

GD4-5

5.0

6.7

5.5

5.0

GD3-17

87.5

91.0

85.5

77.6*

GD3-20

131.6

137.9

129.0

117.0*

Adjusted weight (g)

294.9

298.5

298.0

287.1

Adjusted weight gain (g)

47.8

50.4

47.6

40.7

Gravid uterus weight (g)

83.8

87.5

81.4

76.3*

Corpora lutea (#)

16.0

16.3

15.6

15.3

Implantations (#)

13.9

14.3

13.6

13.3

Pre-implantation loss (%)

12.6

11.6

12.1

12.8

Post-implantation loss (%)

2.5

0.9

1.2

3.8

Live foetuses (#)

13.6

14.2

13.5

12.9

Foetal weight (g)

3.953

4.013

3.859

3.777*

Litter weight (g)

53.4

56.8

51.8

48.7

Placental weight (g)

0.586

0.532

0.562

0.523

Total placental weight (g)

7.779

7.521

7.569

6.684*

Nasal – incomplete ossificationa

14 (6) 8.3%

6 (3) 3.5%

13 (10) 8.2%

19 (13) 15.1%*

*significantly different to controls (p<0.05); **(p<0.01)

a: foetal incidence (litter incidence) foetal incidence (%)

Applicant's summary and conclusion

Conclusions:
Maternal and developmental NOAELs of 1000 mg/kg bw/d can be determined for this study, in the absence of any adverse effects of treatment at the highest dose level.
Executive summary:

Met-THF (2-methyltetrahydrofuran) was administered to groups of 24 time-mated female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats at dose levels of 0 (vehicle control), 100, 300 or 1000 mg/kg bw/d on gestation day (GD) 4-19. The test material was formulated in arachis oil and administered at a constant volume of 4 mL/kg bw. Rats were observed daily for mortality and clinical signs. Bodyweights and food consumption were measured at intervals over the study period.

 

Dams were terminated on GD20, subject to gross necropsy and the uterine contents examined. The gravid uterus and placentae were weighed. All foetuses were sexed, weighed, and assessed for external findings. Approximately half of the foetuses from each litter were assessed for visceral findings following fixation in Bouin’s. The remaining foetuses were assessed for skeletal findings following staining with Alizarin Red S. There was no maternal mortality. Signs of reaction to treatment were limited to post-dose salivation and/or ploughing behaviour in a small number of rats at the highest dos level. Findings are considered to represent a reaction to an irritant or unpalatable test material and are not considered to be adverse. Mean bodyweight initial gain was slightly (but significantly) reduced at 300 and 1000 mg/kg bw/d, but rapidly recovered. Overall weight gain at 1000 mg/kg bw/d was slightly (~11%) but significantly lower than controls; this finding is attributable to the significantly lower gravid uterus weight in this group.

 

Litter parameters were unaffected by treatment. Mean foetal weight was marginally (4.5%) but significantly lower at 1000 mg/kg bw/d. Litter weight in this group was also slightly lower than controls. Total placental weight was significantly lower; however mean placental weight was not significantly lower than controls. The nature and incidence of foetal external, visceral and skeletal parameters was unaffected by treatment.

 

At 1000 mg/kg bw/d, the incidence of foetuses showing incomplete nasal ossification was significantly higher than controls and exceeded the historical control range; however the incidences for the control and 300 mg/kg bw/d groups also exceeded the historical control range. While there was some variation from control for some other ossification parameters at 1000 mg/kg bw/d, there was no obvious effect on the overall pattern of ossification and, therefore, this isolated finding, although supported by slightly lower foetal weight, appears most likely to be incidental and unrelated to treatment.

 

Overall the findings observed for foetuses at 1000 mg/kg bw/d are suggestive of a slight effect on foetal growth although survival and offspring development appeared unaffected. Whether this slight retardation of growth is sufficient to preclude this dose level from being a NOAEL is equivocal and, therefore, the dose level of 1000 mg/kg bw/d is considered to represent the developmental NOAEL. The dose level of 1000 mg/kg bw/d resulted in an initial transient lower body weight gain followed by recovery. Lower body weight gain was again apparent from Day 14 of gestation, but this was influenced by a statistically significant lower contribution from the gravid uterus. There were no accompanying effects apparent on food consumption or clinical condition of the animals. Overall, the extent of effects observed were considered to be insufficient to exclude this dose level from being regarded as a maternal NOAEL.