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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:
2 September to 19 November 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:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: light brown solid

Test animals

Species:
rat
Strain:
other: Sprague-Dawley (Crl:CD(SD))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Quebec, Canada
- Age at study initiation: approximately 87 days
- Weight at study initiation: 222 to 282g
- Fasting period before study: no
- Housing: solid bottom cages with bedding material, 2 or 3 per cage pre-mating, individually after mating
- Diet (ad libitum): PMI Nutrition International LLC Certified Rodent LabDiet 5002
- Water (ad libitum): reverse osmosis-purified on-site drinking water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 to 21.9
- Humidity (%): 38.4 to 57
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 September 2014 To: 3 October 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was placed in a water bath (approximately 28°C) and allowed to melt prior to use. The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 10, 20, 50 mg/mL
- Amount of vehicle: 10mL/kg
- Lot/batch no.: not stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Solubility of the test substance in the vehicle was established in a previous study; therefore, homogeneity assessments were not required. In addition, stability of the test substance in the vehicle at concentrations of 10 and 70 mg/mL was established following 5 and 11 days of room temperature storage.

Samples for concentration analysis were collected from the middle stratum of the first and last preparation of each dosing formulation (including the control group). One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method using flame ionization detection.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until positive evidence of mating
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Day 1 to Day 19 of gestation
Frequency of treatment:
Once daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
25 or 26 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on a previously conducted oral dose range-finding study in pregnant rats which was conducted at dosage levels of 200, 400, 500, and 600 mg/kg/day. In that study, continual maternal toxicity in the 600 mg/kg/day group was evident with reduced mean food consumption, mean body weight gain (body weight loss was found after initial treatment) and lower mean body weights (10% lower than the control group at a maximum on gestation day 5). These effects continued in the 600 mg/kg/day group throughout the treatment period, suggesting continued toxicity to the dam. Maternal toxicity was also evident in the 500 mg/kg/day group with decreases in mean food consumption (up to 43% during the first few days of treatment) and decreases in maternal body weight gain and mean body weights. However, the 500 mg/kg/day group recovered during the second week of exposure and had only 10% reduction in body weight gain over the entire period of gestation. The 500 mg/kg/day dosage level was selected as the high-dosage level for the current study based upon the mild to moderate maternal toxicity observed with transient decreases in food consumption and body weight effects and the minimal (10%) effects on overall body weight gain during treatment period.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before and after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: daily gestational days 0 to 20

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined (g/animal/day) and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: daily gestational days 0 to 20

WATER CONSUMPTION: No
- Time schedule for examinations:

Haematology:
Blood samples for hematology evaluation were collected from all females at the scheduled necropsy. The animals were not fasted overnight prior to blood collection. Blood was collected from the jugular vein, using hand-held restraint, into tubes containing EDTA as the anticoagulant. The following parameters were evaluated:
Total leukocyte count (WBC)
Differential leukocyte count -
Percent
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries. Spleen was weighed and preserved in neutral-buffered 10% formalin for possible further examination.

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 examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, clinical pathology parameters, gravid uterine weights, spleen weights, numbers of corpora lutea, implantation sites, viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Historical control data:
Test Facility historical control data 21 April 1998 to 16 August 2013.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical Observations
Increased incidence of clear material around the mouth were noted in 6 or 9 females receiving 200 or 500 mg/kg/day approximately 1 hour after dosing on gestation days 12 to 18.

Body Weight
At 500 mg/kg/day a transient, statistically significant body weight loss was noted from gestation Days 1 to 3. Lower mean body weight gain was recorded then recorded during gestation days 3 and 4; thereafter body weight gain was comparable with Controls. Overall body mean weight gain during the gestation period was lower at 500 mg/kg/day due to the initial body weight loss/reduced gain and this was considered adverse.

In the 100 and 200 mg/kg/day groups, a statistically significant transient reduction in mean body weight gain was noted after the initial day of dosing (gestation day 1-2). Thereafter, mean body weight gains in these groups were comparable with the control group. The mean body weight and body weight gain were statistically significantly lower in the 200 mg/kg/day group than the control group and were considered adverse. The initial decrements in mean body weight gain in the 100 mg/kg/day group was considered test substance-related, but not adverse because there were no effects on mean absolute body weights during treatment period.

Food Consumption
Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 500 mg/kg/day group was generally lower compared to the control group during gestation days 1-8 and differences were generally statistically significant. Thereafter, mean food consumption was similar to the control group throughout the remainder of gestation. The initial decrements resulted in generally lower mean food consumption during the cumulative gestation days 1-3, 3-6, and 6-9 intervals, and the overall treatment period (gestation days 1-20); differences were generally statistically significant and considered test substance-related and adverse.

Mean food consumption in the 100 and 200 mg/kg/day groups was lower than the control group following the first 2 days of dosing (gestation days 1-3); differences were generally statistically significant. For the remainder of the treatment period, mean food consumption in the 100 and 200 mg/kg/day groups was similar to the control group. The initial lower mean food consumption in the 100 and 200 mg/kg/day groups was considered test substance-related, but not adverse because there was no effect on the overall treatment period, and no corresponding effects on mean absolute body weights.

Haematology
In the 200 and 500 mg/kg/day groups, statistically significantly lower percent eosinophils were noted compared to the control group. However, this value was within the WIL Research historical control data range for non-pregnant females. All other mean hematology values in the test substance-treated groups were similar to the control group; differences were slight, not statistically significant, and/or not present in a dose-related manner.

Necropsy Data
There were no test substance-related findings and spleen weight was unaffected by treatment.

Gravid Uterine Weight
Statistically significantly lower mean gravid uterine weight was noted at 500 mg/kg/day. At 200 mg/kg/day mean gravid uterine weight was higher than the control group and at 100 mg/kg/day was comparable to the control group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Gestation Day 20 Laparohysterectomy Data
The mean litter proportion of postimplantation loss (early resorptions) in the 500 mg/kg/day group was statistically significantly higher when compared to the control group and accounted for all of the total resorptions in this group. A corresponding statistically significant lower mean number and litter proportion of viable fetuses was noted in this group when compared to the control group. The percentage of female fetuses and mean female fetal weights were also lower compared to the concurrent control group; the difference in female fetal weights being statistically significant (p<0.05). The percentage of female fetuses was lower than the WIL Research historical control data range. Based on these results, the test substance appears to exhibit a more pronounced effect on female fetuses and it is likely the lower number of female fetuses could be due to female implantations having a higher resorption rate than the male fetuses at 500 mg/kg/day. These effects on intrauterine growth and survival at 500 mg/kg/day were considered test substance-related and adverse.

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 100 and 200 mg/kg/day.

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Fetal Morphology Data
The numbers of fetuses (litters) available for morphological evaluation were 371(25), 329(22), 354(23), and 285(22) in the control, 100, 200, and 500 mg/kg/day groups, respectively. Malformations were observed in 1(1), 0(0), 1(1), and 6(6) fetuses (litters) in the respective dose groups.

The mean litter proportion of total malformations and variations in the 500 mg/kg/day group were higher than the control group due to higher mean litter proportions of external malformations and skeletal variations; the differences in skeletal and total variations were statistically significant, while the mean litter proportions of external and total malformations exceeded the maximum mean values in the WIL Research historical control data. These higher values were attributed to test substance-related external malformations (vertebral agenesis, exencephaly without open eyelid, anophthalmia, facial cleft, omphalocele, body shorter than normal, tarsal flexure, and microcephaly), and skeletal variations (7th cervical rib) noted at 500 mg/kg/day. There were no test substance-related external, visceral, or skeletal malformations or variations noted in the 100 and 200 mg/kg/day groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Results of Concentration Analysis

 

Mean Concentration mg/mL (% of target)

Date of Preparation

Group 2
(10 mg/mL)

Group 3
(20 mg/mL)

Group 4
(50 mg/mL)

09 Sept 2014

9.96 (99.6)

19.9 (99.5)

50.1 (100)

24 Sept 2014

9.66 (96.6)

18.4 (92.2)

48.0 (96.0)

Table 2: Summary of Body Weight Changes During Gestation (g)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

13
3.1
25

8**
3.2
22

6**
4.4
23

-16**
7.7
22

3-6

Mean
SD
N

12
5.0
25

15
5.6
22

13
5.3
23

6**
11.1
22

6-9

Mean
SD
N

15
4.2
25

12
3.9
22

13
4.6
23

20**
8.4
22

9-12

Mean
SD
N

19
5.3
25

18
7.8
22

17
5.7
23

23
6.3
22

12-15

Mean
SD
N

19
5.2
25

21
8.8
22

19
5.2
23

17
5.6
22

15-20

Mean
SD
N

71
10.4
25

73
9.2
22

72
10.5
23

65
13.9
22

1-20

Mean
SD
N

148
17.9
25

146
13.6
22

141
19.9
23

115**
23.8
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Mean differences calculated from individual differences

Nongravid weight(s) not included in calculation of mean

Table 3: Summary of Gravid Uterine Weights and Net Body Weight changes (g)

Group (mg/kg/day)

 

Control

100

200

500

Initial body weight

Mean
SD
N

253
13.9
25

252
14.0
22

250
13.8
23

251
12.0
22

Terminal body weight

Mean
SD
N

406
26.2
25

404
18.2
22

398
20.9
23

373**
27.6
22

Gravid uterine weight

Mean
SD
N

85.8
12.55
25

86.2
12.73
22

91.7
12.19
23

75.2**
20.67
22

Net body weight

Mean
SD
N

320.6
19.43
25

317.9
15.48
22

306.7*
17.09
23

297.6**
13.92
22

Net body weight change

Mean
SD
N

68
12.86
25

65.8
13.00
22

56.5**
15.62
23

47.0**
9.57
22

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

Table 4: Summary of Food consumption During Gestation (g/animal/day)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

19
2.1
25

17**
1.6
22

16**
1.8
23

8**
3.0
22

3-6

Mean
SD
N

20
2.0
25

19
1.5
22

19
1.8
23

12**
3.8
22

6-9

Mean
SD
N

21
2.2
25

20
1.8
22

20
2.0
23

19**
2.1
22

9-12

Mean
SD
N

23
1.8
25

22
1.4
22

22
1.9
23

22
1.3
22

12-15

Mean
SD
N

23
1.8
25

23
2.0
22

23
2.3
23

23
2.3
22

15-20

Mean
SD
N

25
2.0
25

25
1.9
22

25
2.3
23

25
2.4
22

1-20

Mean
SD
N

22
1.6
25

21
1.3
22

21
1.6
23

20**
1.7
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Nongravid weight(s) not included in calculation of mean

Table 5: Summary of Food Consumption During Gestation (g/kg/day)

Group (mg/kg/day)

 

Control

100

200

500

Day

 

 

 

 

 

1-3

Mean
SD
N

72
7.1
25

64**
4.4
22

61**
6.7
23

31**
11.2
22

3-6

Mean
SD
N

73
5.2
25

70
4.2
22

70
6.3
23

49**
13.8
22

6-9

Mean
SD
N

74
5.9
25

70
5.0
22

72
6.6
23

75
7.6
22

9-12

Mean
SD
N

74
4.2
25

72
3.6
22

73
6.2
23

80**
4.9
22

12-15

Mean
SD
N

71
6.0
25

71
5.6
22

73
6.5
23

77**
7.5
22

15-20

Mean
SD
N

68
4.6
25

68
4.2
22

69
4.9
23

75**
5.6
22

1-20

Mean
SD
N

71
4.1
25

69
3.2
22

70
4.4
23

68
4.6
22

** = Significantly different from the control group at 0.01 using Dunnett's test

Nongravid weight(s) not included in calculation of mean

Table 6: Summary of Fetal Data at Scheduled Necropsy

Dose
mg/kg/day

 

sex

Viable fetuses

Dead fetuses

Resorptions

Post implantation loss

Implantation sites

Corpora lutea

Pre implantation loss

Fetal weight (g)

No. of gravid females

M

F

early

late

0
(control)

Total
mean
SD
SE

198
7.9
1.98
0.40

173
6.9
2.10
0.42

371
14.8
2.29
0.46

 0
 0.0
 0.00
 0.00

14
0.6
0.65
0.13

 0
 0.0
 0.00
 0.00

14
0.6
0.65
0.13

385
15.4
2.38
0.48

422
16.9
2.09
0.42

37
1.5
2.26
0.45

NA
3.7
0.17
0.03

25

100

Total
mean
SD
SE

165
7.5
2.35
0.50

164
7.5
2.24
0.48

329
15.0
2.50
0.53

 0
 0.0
 0.00
 0.00

30
1.4
2.08
0.44

 0
 0.0
 0.00
 0.00

30
1.4
2.08
0.44

359
16.3
2.01
0.43

384
17.5
1.90
0.40

25
1.1
1.73
0.37

NA
3.8
0.22
0.05

22

200

Total
mean
SD
SE

174
7.6
2.11
0.44

180
7.8
2.12
0.44

354
15.4
2.02
0.42

 0
 0.0
 0.00
 0.00

14
0.6
0.78
0.16

 1
 0.0
 0.21
 0.04

15
0.7
0.88
0.18

369
16.0
1.82
0.38

404
17.6
2.90
0.61

35
1.5
3.29
0.69

NA
3.9
0.29
0.06

23

500

Total
mean
SD
SE

166
7.5
2.60
0.55

119
5.4
1.92
0.41

285
13.0*
3.58
0.76

 0
 0.0
 0.00
 0.00

36
1.6
1.47
0.31

 0
 0.0
 0.00
 0.00

36
1.6
1.47
0.31

321
14.6
3.40
0.73

369
16.8
4.08
0.87

48
2.2
2.87
0.61

NA
3.7
0.47
0.10

22

* significantly different from Control group at 0.05

Table 7: Test Substance-Related External Malformations

 

Absolute no. (% per litter)

WIL HC Mean
(range; % per litter)

Dose (mg/kg/day)

Control

100

200

500

Vertebral Agenesis

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.4)

Exencephaly without open eyelid

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.3)

Right anophthalmia

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.6)

0.0 (0.0-0.6)

Facial cleft

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.3)

Omphalocele

0 (0.0)

0 (0.0)

0 (0.0)

2 (0.6)

0.0 (0.0-0.3)

Body shorter than normal

0 (0.0)

0 (0.0)

0 (0.0)

1 (0.3)

0.0 (0.0-0.0)

Tarsal flexure

0 (0.0)

0 (0.0)

0 (0.0)

2 (4.9)

0.0 (0.0-0.3)

Microcephaly

0 (0.0)

0 (0.0)

0 (0.0)

1 (4.5)

0.0 (0.0-0.0)

HC = historical control

Applicant's summary and conclusion

Conclusions:
Administration of tetrahydrothiophene 1,1-dioxide to pregnant Sprague Dawley (Crl:CD(SD)) rats at 0, 100, 200 or 500 mg/kg/day resulted in lower mean body weights, mean body weight changes, and food consumption at 500 mg/kg/day; lower body weights and decreased body weight change were also noted in the 200 mg/kg/day group. These changes at 200 and 500 mg/kg/day were considered to be adverse.

In addition at 500 mg/kg/day, a higher mean litter proportion of postimplantation loss (early resorptions) and corresponding lower mean litter proportion of viable fetuses, a lower percentage of female fetuses and lower female fetal weights, and increased incidences of fetal external malformations were noted which were all considered to be adverse.

Therefore 200 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for embryo/fetal development and based on the effects on body weight and food consumption the maternal NOAEL was considered to be 100 mg/kg/day.