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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:
2017-04-27 to 2017-08-15
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)
Deviations:
yes
Remarks:
Deviations were not considered to have affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V. (Kreuzelweg 53, 5961 NM Horst, Netherlands)
- Age at study initiation: 11-15 weeks (males and females)
- Weight at study initiation: 196-275 grams (females)
- Fasting period before study: No
- Housing: Females: Acclimatization period (maximum 5 animals/cage) Makrolon type IV cages; Pregnancy (individually) Makrolon type-III cages
Males: Acclimatization and pairing (individually) Makrolon type-III cages
- Diet (e.g. ad libitum): Powdered standard VRF1 rat/mouse maintenance diet ad libitum (supplied by Special Diet Services).
- Water (e.g. ad libitum): Tap water in bottles ad libitum.
- Acclimation period: 5-7 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light (7:00 am to 7:00 pm) / 12 hours dark (7:00 pm to 7:00 am).

IN-LIFE DATES: From: 2017-04-20 To: 2017-05-24

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets prepared on 28 April and 22 May 2017. Diet prepared for Groups 2 and 4 on 31 May 2017.
- Mixing appropriate amounts with (Type of food): The test item was incorporated into the basal diet to provide the required concentrations by dilution of an appropriate premix. The required amount of test item was added to an approximately equal amount of sieved diet and stirred. An amount of sieved diet approximately equal to the weight of the mixture was added and the mixture stirred until it appeared homogeneous. This doubling-up process with sieved diet continued until approximately half of the premix diet had been added. At this stage, the mixture was ground using a mechanical grinder. Coarse diet was then added to make the premix up to the required amount and mixing continued in a Turbula mixer for 100 revolutions at 16 rpm. This ensured the test item was dispersed in the diet. An aliquot of this premix was then diluted with further quantities of coarse basal diet to prepare the required concentration for each test group.
- Storage temperature of food: Diets prepared on 28 April and 22 May 2017 were provided frozen (in dry ice). These diets were stored in a freezer and the required amount of diet was removed for feeding on each occasion. Diet prepared for Groups 2 and 4 on 31 May 2017 was provided at ambient temperature as it was administered within 15 days of preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The method of analysis used was that validated in Envigo Study Number JW52PN (GLP) Validation of an Analytical Method and Determination of Homogeneity and Stability in VRF1 Diet Formulations. Each analytical batch included the concurrent preparation and analysis of three QCs, one at each of the three nominal dietary concentrations. Each batch was calibrated using matrix matched calibration standards.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: After acclimatization, females were housed with sexually mature males in a cage for a period approximately of 16 hours
- M/F ratio per cage: Maximum 2:1
- Length of cohabitation: approximately of 16 hours
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: yes (Male rats of the same source and strain were used for mating only)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
a) Mating and consequently vaginal smears were discontinued when considered necessary for internal workload distribution. This had no repercussions on the study, as gestation (study period) started once mating was successful.
b) Female no. 114 was assigned to a treatment group on day 2 of the study, instead of “each mating time” as indicated in the study plan. The reason for that was to have the updated results of pregnant females and assign this female taking these results into consideration.
Duration of treatment / exposure:
Females received the test item from days 3 to 19 post coitum
Frequency of treatment:
Continuously by dietary admixture
Duration of test:
Day 3 to Day 19 post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Group 1 (Control)
Dose / conc.:
2 500 ppm
Remarks:
Group 2
188.2 mg/Kg/day
Dose / conc.:
5 000 ppm
Remarks:
Group 3
369.3 mg/Kg/day
Dose / conc.:
10 000 ppm
Remarks:
Group 4
908.2 mg/Kg/day
No. of animals per sex per dose:
Group 1: 22 females
Group 2: 23 females
Group 3: 23 females
Group 4: 24 females

15 males ordered and 10 males from Envigo CRS S.A.U. stock
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary levels were selected based on a dose-range finding toxicity study in rats (Envigo non-GLP Study RH75LP Dose Range Finder for Prenatal Developmental Toxicity in Rats), using dietary levels of 2000, 5000 and 10000 ppm. For the GLP study, the same high dietary exposure level was selected, taking into account the mild signs of maternal and developmental toxicity observed in the preliminary DRF study. For intermediate and low dietary levels, a two-fold interval is considered optimal for setting the descending levels.
- Rationale for animal assignment (if not random): Rats were assigned to the different groups at random in all groups each mating time. An acceptable distribution of males to which the females were mated was ensured.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs for signs of reaction to treatment and/or symptoms of ill health during days 3-20 of pregnancy. For male rats clinical signs were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and daily from day 3 to 20 post coitum. For male rats, body weights were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post coitum)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day #20 post coitum
- Organs examined: At the scheduled necropsy on day 20 post coitum, females were sacrificed by CO2 inhalation and the foetuses removed by caesarean section.
Post-mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of foetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females were weighed during necropsy on day 20 post coitum to enable the calculation of the corrected body-weight gain.

When no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulphide (Salewski, 1964) to accentuate possible haemorrhagic areas of implantation sites.
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: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [see Table 7.]
Statistics:
The following comparisons were performed:
Group 1 vs. Groups 2, 3 and 4 to analyse food consumption, body weights and reproduction data: Means and standard deviations of various data were calculated.

For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level. Treated groups were compared to control using Williams' test (Williams, 1971, 1972), unless there was evidence against a monotonic dose-response when Dunnett's test (Dunnett, 1955, 1964) was performed instead.

A non-parametric analysis was performed if Bartlett's test (Bartlett, 1937) was still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test (Shirley, 1977), unless there was evidence against a monotonic dose-response when Steel's (Steel, 1959) test was performed instead.

Cochran and Cox test has been used directly by the Pristima system in order to analyse reproductive data.

Dunnett or Cochran-Armitage chi square for numerical and categorical statistical tests were directly used by the Pristima system to analyse body weight gain.

Significant differences between the groups compared were expressed at the 5% (p ≤0.05) or 1% (p ≤0.01) level.
Historical control data:
Envigo Historical Control Data provided - HCD Prenatal Developmental Toxicity in SD Rats (Annex 5 of attached study report)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hunched posture was occasionally observed in some animals exposed to 5000 ppm (females no. 64 and 72 on days 4 and 6-7 of gestation, respectively) and to 10000 ppm (females no. 91 and 95 on days 12-14 and 4, respectively).

No clinical signs were recordedin the animals exposed to 2500 ppm or in the Control group.
Mortality:
no mortality observed
Description (incidence):
No mortality was recorded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no relevant effect on mean body weights. However, statistically significant lower body weight gain relative to day 3 of treatment was observed in animals exposed to 10000 ppm between days 4 and 10 (between -23% and 77% with respect to Control), and in animals exposed to 5000 ppm on days 4, 6 and 7 (between 30% and 57% with respect to Control). For each of these observations, body weight gain was lower than in the Control group. No relevant differences were observed in body weight gain between groups from day 15 onwards.

Although a statistically significant effect on body weight gain between days 4 and 10 was observed at 10000 ppm, it was considered non-adverse, since it was a transient effect and no relevant differences were observed in body weight gain later during gestation, nor were there any effects on the general well-being of the dams in this group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was an increase in the food consumption in the females exposed to 10000 ppm. The differences observed when compared with the Control group ranged from 14% to 35% between days 3 and 18 of exposure and were statistically significant.

No effect was observed at the 2500 or 5000 ppm exposure levels.
Food efficiency:
not specified
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:
no effects observed
Description (incidence and severity):
No findings were recorded in the macroscopic examination at hysterectomy in the rats treated with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical Signs
Hunched posture was occasionally observed in some animals exposed to 5000 ppm (females no. 64 and 72 on days 4 and 6-7 of gestation, respectively) and to 10000 ppm (females no. 91 and 95 on days 12-14 and 4, respectively). No clinical signs were recorded in the animals exposed to 2500 ppm or in the Control group.

Body weight and weight changes
There was no relevant effect on mean body weights. However, statistically significant lower body weight gain relative to day 3 of treatment was observed in animals exposed to 10000 ppm between days 4 and 10 (between -23% and 77% with respect to Control), and in animals exposed to 5000 ppm on days 4, 6 and 7 (between 30% and 57% with respect to Control). For each of these observations, body weight gain was lower than in the Control group. No relevant differences were observed in body weight gain between groups from day 15 onwards.

Food consumption and compound intake
There was an increase in the food consumption in the females exposed to 10000 ppm. The differences observed when compared with the Control group ranged from 14% to 35% between days 3 and 18 of exposure and were statistically significant. No effect was observed at the 2500 or 5000 ppm exposure levels.

Maternal developmental toxicity

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 number of pregnant:
no effects observed
Description (incidence and severity):
There were no test-item related changes.

The summary of performance of mated females is provided in Table 2.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Slight differences were recorded in some reproductive parameters. Although statistically significant differences in placenta weight were observed at the 10000 ppm exposure level, this was not considered toxicologically significant, as the ratio of mean foetal/placental weights per dam were similar among all study groups, and ranged from 5.82 to 6.05 and means were only 8% lower than in the Control group.

There were no differences between the left and the right side of the uterine horns in the distribution of resorptions or in the total resorptions in the uterus. However, mean uterus weight was 6% lower at 10000 ppm compared with the Control group (not statistically-significant), which may have been related to the statistically significant lower foetal body weight in group 4.

No differences with respect to Control were recorded at the 2500 or 5000 ppm exposure levels.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant lower mean male/female foetal body weights were observed in the 10000 ppm exposure level. Differences were 6 and 5% lower, respectively, when compared to Control. However, this was not considered an adverse effect, since the magnitude of change was small, in the absence of any structural abnormalities, and the mean values per litter were within the Historical Control range of foetal body weights observed in developmental toxicity studies with Sprague-Dawley rats conducted at Envigo CRS, S.A.U.

No differences were recorded among foetuses from 2500 or 5000 ppm exposure levels.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No differences in the ratio of male/female foetuses were noted.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant lower mean litter weights were observed in the 10000 ppm exposure level. Differences were 6% lower, when compared to Control. However, this was not considered an adverse effect, since the magnitude of change was small, in the absence of any structural abnormalities, and the mean values per litter were within the Historical Control range of foetal body weights observed in developmental toxicity studies with Sprague-Dawley rats conducted at Envigo CRS, S.A.U.

No differences were recorded among foetuses from 2500 or 5000 ppm exposure levels.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings which could be attributed to test item exposure were observed.

Group 3: 5000 ppm (Litter no. 65: One male foetus (no. 14) presented whole filamentous and vestigial tail).
Group 4: 10000 ppm (Litter no. 98: One male foetus (no. 12) had generalized oedema)
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test item related alterations were recorded.

Group 3: 5000 ppm (Litter no. 75: One female foetus (no. 8) showed short/bent/thickened and incompletely ossified humerus, radius and ulna and bent/incompletely ossified scapula). No other major abnormalities were observed in the rest of the foetuses examined.

At 10000 and 2500 ppm there was an increased incidence of short supernumerary 14th ribs and delayed/incompletely ossification/unossified sternebrae, compared to the concurrent Control. However, the incidence at 5000 ppm was similar to that observed in the Control group. Furthermore, the incidence of delayed/incompletely ossification/unossified sternebrae at 10000 and 2500 ppm was within the Historical Control range in developmental toxicity studies with Sprague-Dawley rats conducted at Envigo CRS, S.A.U. Consequently, these findings were not considered related to test item exposure.

The incidence and/or distribution of major and minor abnormalities and skeletal variants among groups showed no relationship to test item exposure.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some isolated major abnormalities in visceral examination were recorded in a total of four foetuses.

Control group: Litter no. 18, foetus no. 6 (female): incomplete aortic arch and dilated pulmonary trunk

2500 ppm: Litter no. 38, foetus no. 2 (male): muscular ventricular septal defect

5000 ppm: Litter no. 65, foetus no. 14 (female): abdominal spinal cord termination and imperforate anus. This abnormal finding also correlated with the external observation (threadlike tail).

10000 ppm: Litter no. 105, foetus no. 2 (male): inguinal hernia

As no exposure-effect relationship was observed, and taking into account the low incidence, these findings were not considered to be attributable to test item exposure. In addition, visceral abnormalities were observed in all groups, including the Control.
Details on embryotoxic / teratogenic effects:
Foetal body weight change and changes in Litter size and weights
Statistically significant lower mean litter and male/female foetal body weights were observed in the 10000 ppm exposure level. Differences were 6, 6 and 5% lower, respectively, when compared to Control. However, this was not considered an adverse effect, since the magnitude of change was small, in the absence of any structural abnormalities, and the mean values per litter were within the Historical Control range of foetal body weights observed in developmental toxicity studies with Sprague-Dawley rats conducted at Envigo CRS, S.A.U.

No differences were recorded among foetuses from 2500 or 5000 ppm exposure levels.

Visceral Malformations
Some isolated major abnormalities in visceral examination were recorded in a total of four foetuses.

As no exposure-effect relationship was observed, and taking into account the low incidence, these findings were not considered to be attributable to test item exposure. In addition, visceral abnormalities were observed in all groups, including the Control.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 2. Food consumption - group mean values (g/animal/day)

Group/Sex

 

Day

0-3

3-6

6-9

9-12

12-15

15-18

18-20

Statistics Test

Sh

Sh

Sh

Sh

Sh

Sh

Sh

Sh

1F

Mean

19

20

21

21

21

24

24

SD

6.1

5.1

4.7

5.3

4.6

5.3

2.9

N

21

21

21

21

21

21

21

 

2F

Mean

18

20

19

21

20

24

23

SD

1.8

1.7

1.6

2.4

1.9

2.1

2.6

N

19

19

19

19

19

19

18

% of 1F

92

97

91

99

96

100

98

 

3F

Mean

19

22

21

22

21

24

24

SD

2.7

5.4

3.4

4.0

2.1

1.9

3.7

N

20

20

20

20

20

20

20

% of 1F

99

112

96

105

102

101

100

 

4F

Mean

20

27**

24*

26**

26**

28**

25

SD

2.3

9.7

5.9

7.0

7.1

7.2

6.9

N

23

23

23

23

23

23

23

% of 1F

102

133

113

126

128

117

107

Sh - Treated groups compared with Control using Shirley’s test

* p ≤0.05

** p ≤0.01

Table 3. Body weight gain - group mean values - % respect to Day 3

Group/Sex

 

Day

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Statistics Test

 

 

Du

Du

CA

CA

Du

CA

Du

 

 

 

 

 

 

 

 

 

 

1F

Mean

0.00

1.46

2.86

4.15

5.48

6.69

9.01

11.31

12.50

13.74

15.30

18.39

20.92

26.30

32.65

38.65

45.47

51.91

SD

0.000

2.021

2.554

3.377

2.975

3.292

3.485

3.577

3.946

4.078

4.332

4.494

5.368

5.859

6.918

8.119

9.284

9.097

N

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

 

2F

Mean

0.00

2.05

3.20

4.24

5.44

6.86

8.77

11.25

12.76

14.06

15.87

18.13

20.66

26.08

31.68

38.29

44.15

50.62

SD

0.000

2.303

2.030

1.661

1.680

2.002

2.016

2.255

2.530

2.956

3.076

3.259

3.509

4.581

5.111

6.680

7.191

8.996

N

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

% of 1F

100

140

112

102

99

103

97

100

102

102

104

99

99

99

97

99

97

98

 

3F

Mean

0.00

-0.44**

1.36

1.79*

3.13*

5.77

7.63

10.42

12.52

14.48

16.17

18.92

21.79

26.42

32.74

39.12

45.35

51.73

SD

0.000

1.506

2.190

3.492

3.908

2.142

2.059

1.984

2.332

2.538

2.805

2.969

2.836

3.218

3.900

4.166

4.239

5.120

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

% of 1F

100

-30

48

43

57

86

85

92

100

105

106

103

104

100

100

101

100

100

 

4F

Mean

0.00

-1.79**

-0.16**

0.82**

2.25**

4.38*

5.95**

8.73**

10.99

11.84

14.05

16.13

19.42

24.35

29.28

35.18

40.73

46.51

SD

0.000

2.165

1.694

2.340

2.020

2.778

3.061

2.861

3.049

4.012

5.288

6.217

4.452

5.424

5.608

6.219

7.614

8.039

N

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

% of 1F

100

-123

-6

20

41

65

66

77

88

86

92

88

93

93

90

91

90

90

CA Treated groups compared with Control Trend test using Cochran Armitage test

Du Treated groups compared with Control using Dunnett’s test

* p ≤0.05

** p ≤0.01

Table 4. Summary of Performance of Mated Females

Group

1

2

3

4

Dietary Exposure Levels (ppm)

0

2500

5000

10000

Number of mated females

22

23

23

24

Number of pregnant females

21

19

20

23

The following females were not pregnant:

Group 1: 14

Group 2: 34, 35. 45 and 46

Group 3: 61, 81 and 83

Group 4: 104

Table 5. Summary of Female Reproduction Data with Mean Foetal Data by Group

Parameter

Dose Group

Control

2500 ppm

5000 ppm

10000 ppm

Corpora Lutea

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

17.1

15.9

16.4

16.9

SDevs

2.07

2.16

1.98

1.94

 

Implantation Sites

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

16.1

14.5

15.3

15.7

SDevs

2.70

3.45

1.74

3.19

 

Preimplantation Loss

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

1.0

1.4

1.1

1.2

SDevs

1.67

2.14

2.05

1.83

 

Preimplantation Loss %

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

6.0

9.6

6.0

8.2

SDevs

9.87

18.51

10.82

15.39

 

Early Resorption

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

1.2

0.7

0.6

0.8

SDevs

1.76

0.75

0.69

1.03

 

Late Resorption

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

0.0

0.0

0.0

0.0

SDevs

0.00

0.00

0.00

0.00

 

Total Resorption

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

1.2

0.7

0.6

0.8

SDevs

1.76

0.75

0.69

1.03

 

Foetal Sex Females

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

7.5

6.5

7.4

7.8

SDevs

2.44

2.52

2.37

2.83

 

Foetal Sex

Hermaphrodite

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

0.0

0.0

0.0

0.0

SDevs

0.00

0.00

0.00

0.00

 

Foetal Sex Males

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

7.4

7.3

7.4

7.0

SDevs

2.48

2.19

2.30

2.42

 

Foetal Sex Undetermined

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

0.0

0.0

0.0

0.0

SDevs

0.00

0.00

0.00

0.00

 

Male Percent

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

49.6

55.1

50.0

47.4

SDevs

10.68

15.73

14.52

14.29

 

Dead Foetuses

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

0.0

0.0

0.0

0.0

SDevs

0.00

0.00

0.00

0.00

 

Live Foetus

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

14.9

13.8

14.7

14.8

SDevs

3.53

3.3

2.08

3.10

 

Post Implantation Loss

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

1.2

0.7

0.6

0.8

SDevs

1.76

0.75

0.69

1.03

 

Post Implantation Loss

%

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

8.4

4.4

3.9

5.0

SDevs

12.39

4.88

5.08

6.40

 

Uterus Weight

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

82.439

77.293

79.995

77.432

SDevs

17.8002

17.2467

9.0109

15.7047

 

Foetal Weight Dam

Mean

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

3.487

3.522

3.471

3.291*D

SDevs

0.2402

0.2101

0.2190

0.2213

 

Placenta Weight Dam

Mean

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

0.597

0.605

0.574

0.548*C

SDevs

0.0678

0.0726

0.0879

0.0448

 

Male Foetal Mean

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

3.590

3.594

3.555

3.379*D

SDevs

0.2566

0.2383

0.2402

0.2477

 

Female Foetal Mean

p-value

-

-

-

-

N

21@

18@

20@

23@

Mean

3.379

3.428

3.390

3.222*D

SDevs

0.2146

0.1781

0.2206

0.2249

 

Litter Weight

p-value

-

-

-

-

N

21@

19@

20@

23@

Mean

51.466

48.421

50.773

48.598

SDevs

11.2743

11.2617

6.1096

10.3335

@= Number examined reduced due to excluded data

*D = Dunnett LSD Test Significant at the 0.05 level

*C = Cochran and Cox Test Significant at the 0.05 level

Table 6. Fetal Body Weight

Group

Male and female foetal weight

per litter (g)

Male foetal weight

per litter (g)

Female foetal weight

per litter (g)

0 ppm

3.487

3.590

3.379

2500 ppm

3.522

3.594

3.428

5000 ppm

3.471

3.555

3.390

10000 ppm

3.291*

3.379*

3.222*

Historical Control dataa

Mean 3.66

Range 2.9 to 4.4

Mean 3.77

Range 3.2 to 4.4

Mean 3.56

Range 2.6 to 4.3

  a In seven studies conducted between 2010 and 2014, control rats of the same strain housed in similar environmental conditions.

    Envigo Historical Control Data included in Annex 5 of the study report.

Table 7. Summary of Foetal Examination Data

 

Dosage Group

Control

2500 ppm

5000 ppm

10000 ppm

Number of Dams/Foetus:

22/312

23/263

23/294

23/341

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Head

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Tail

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Whole

 

0/0

0/0

1/1

0/0

% Litter

0

0

5

0

% Foetal

0.00

0.00

0.28

0.00

 

Skin

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Limb/Paw

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Thorax

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

Abdomen/Trunk

Number Examined Litter/Foetus:

21/312

19/263

20/292

23/341

 

General Comments

Number Examined Litter/Foetus:

14/209

15/207

17/250

16/218

 

Oedema

 

0/0

0/0

0/0

1/1

% Litter

0

0

0

6

% Foetal

0.00

0.00

0.00

0.89

 

 

Table 8. Skeletal Examination

Group

Number of 14thribs supernumerary

Delayed/incompletely ossification/unossified sternebrae

0 ppm

8%

9%

2500 ppm

20%

21%

5000 ppm

8%

10%

10000 ppm

22%

21%

Historical Control Dataa

foetal incidence (% of total examined)

range of HC foetal incidence per study

 

9%

Range 4 to 17%

 

17%

Range 7 to 29%

a In four studies conducted between 2011 and 2014, control rats of the same strain housed in similar environmental conditions. Envigo Historical Control Data is included in Annex 5.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 10000 ppm was determined to be the maternal No Observed Adverse Effect Level (NOAEL) for pregnant females.

With respect to the effects on embryofoetal development, 10000 ppm was determined to be the No Observed Adverse Effect Level (NOAEL).
Executive summary:

In a key Guideline (OECD 414) pre-natal developmental toxicity study, the test material (Rosin, fumarated; CAS# 65997-04-8) was administered by continuous dietary admixture to three groups of Sprague-Dawley rats, between gestation days 3 and 19 (inclusive) at dietary concentrations of 2500, 5000, and 10000 ppm (equivalent to mean achieved dosages of approximately 0, 188.2, 369.3 and 908.2 mg/Kg bw/day). An additional group of female rats was treated with basal laboratory diet to serve as a control.

 

Clinical signs, body weight change, and food consumption was monitored during the study. All females were terminated on gestation day 20 and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weights, sex and external and internal macroscopic appearances were recorded. Half of the pups of each litter were examined for detailed

skeletal development and the remainder were subjected to detailed visceral examination.

 

No mortality and no clinical signs were apparent for adult females throughout the study at dietary concentrations of 2500, 5000 or 10000 ppm of the test material. Hunched posture was transiently observed in a few of the animals exposed to the test material at 5000 or 10000 ppm, but was not considered to be a toxic effect since the incidence was unrelated to exposure concentration.

 

Food consumption in animals exposed at 10000 ppm was higher than the control group during the exposure period. Despite there being no effect on absolute body weight, a trend to lower body weight gain was observed in the animals exposed at 10000 ppm, especially between days 4 and 10 of gestation. However, this trend was not considered toxicologically relevant since it was a transient effect and no relevant differences were observed in body weight gain later during gestation, nor were there any effects on the general well-being of the dams in this group.

 

There were no relevant findings in the observations derived from hysterectomy. However, slightly lower mean foetal body weights at the10000 ppm exposure level (compared to control) could help explain the slightly lower placenta and uterus weights observed at this exposure level, as was evident when mean placental or uterus weights were compared to foetal weights. There was no effect on the sex ratio post exposure to the test material.

 

Gross necropsy did not reveal any remarkable findings.

 

A low incidence of major foetal abnormalities was observed at 10000, 5000 and 2500 ppm and in the control group (1/312 in Control, 1/263 at 2500 ppm, 2/294 at 5000 ppm and 2/341 at 10000 ppm). These were considered to be incidental, given their incidence and distribution across the treatment and control groups.

 

Skeletal examination of the foetuses showed a slightly increased incidence of delayed/incomplete ossification/unossified sternebrae in the high and low exposure groups (9% foetuses in Control, 21% at 2500 ppm, 10% at 5000 ppm and 21% at 10000 ppm) and short supernumerary 14thribs (8% of foetuses in Control, 20% at 2500, 8% at 5000 and 22% at 10000 ppm). However, the incidences in the high and low exposure groups were similar. Furthermore, the incidence of delayed/incompletely ossification/unossified sternebrae at 10000 and 2500 ppm was within the Historical Control range. Given the distribution of these findings within the treatment groups, they were not considered adverse or treatment-related.

 

Skeletal and visceral examination of the foetuses showed the presence of major abnormalities in one foetus at 10000 ppm (litter no. 105, foetus no. 2), two at 5000 ppm (litter no. 65, foetus no. 14 and litter no. 75, foetus no. 8), one at 2500 ppm (litter no. 38, foetus no. 2) and one in the control group (litter no. 18, foetus no. 6). Given their incidence and distribution, these abnormalities were considered unrelated to treatment with the test material.

 

Based on the results of this study, 10000 ppm was determined to be the maternal No Observed Adverse Effect Level (NOAEL) for pregnant females. Although a statistically significant effect on body weight gain between days 4 and 10 was observed at 10000 ppm, it was considered non-adverse, since it was a transient effect and no relevant differences were observed in body weight gain later during gestation, nor were there any effects on the general well-being of the dams in this group.

 

With respect to effects on embryofoetal development, 10000 ppm was determined to be the NOAEL. Although slight but statistically significant reduced foetal body weights were observed, this was not considered an adverse effect, due to the small magnitude of the change and because the mean value was within the Historical Control range. Despite some abnormalities in all dietary exposure groups as well as in control foetuses, there was no evidence that dietary exposure to the test material at 10000 ppm increased the occurrence of teratogenic effects. This dose was therefore considered to be the NOAEL.