Registration Dossier

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:
2015-08-13 to 2015-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2015-08-13 to 2015-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across source
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were detected.

One female in the 15000 ppm dietary exposure group had red staining around the ano-genital region on Day 11 of gestation. In isolation thiswas considered incidental and unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, body weight gain was generally lower than control throughout gestation, with differences frequently attaining statistical significance and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by dietary exposure to 3000 or 7500 ppm of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, food consumption was generally lower than control between gestation days 3 and 11, with differences attaining statistical significance. A slight recovery in food consumption was evident thereafter.

Food consumption during gestation was unaffected by dietary exposure to 3000 or 7500 ppm
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were detected during the macroscopic examination of the pregnant females at termination on gestation day 20.
Pre- and post-implantation loss:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Details on maternal toxic effects:
Maternal dietary exposure to 3000, 7500 or 15000 ppm of the test item resulted in no effects on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and postimplantation losses. At the 15000 ppm dietary exposure level, placental weights were statistically significantly reduced.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: effects on body weight and food consumption at the highest concentration tested.
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, mean foetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance.

At the 3000 and 7500 ppm dietary exposure levels, mean male foetal weights and mean foetal weights (both sexes combined) were statistically significantly lower than control. All individual mean litter values were within historical background control ranges (mean male foetal weight: 3.24-5.03g and mean foetal weight: 2.42-4.97g) and in isolation at these levels were considered to reflect normal biological variation.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor the distribution of findings observed during external examination of the foetuses at necropsy on gestation day 20 indicated any adverse effect of maternal dietary exposure on foetal development.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm exposure level, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of foetuses/litters increased compared with control and differences frequently attaining statistical significance. These parameters included incomplete ossification of the frontal, parietal, interparietal, occipital, squamosal, jugal, zygomatic process of maxilla and squamosal, premaxilla bones of the skull, no ossification of the frontal and presphenoid bones of the skull, incomplete ossification of the lumbar (neural) arch, incomplete/no ossification of the sacral (neural) arch, less than 4 caudal vertebrae ossified, no ossification of the sternebra, partially split xiphoid cartilage,wavy/thickened/incomplete ossification of the ribs, and no ossification/incomplete ossification of the pubis, and metacarpals. There was also a lower incidence of foetuses showing no ossification of the squamosal bone of the skull and ossification centre associated with 1st lumbar vertebra.

At the 3000 and 7500 ppm dietary exposure levels, the incidence and type of skeletal findings observed did not indicate any adverse effect of maternal treatment. The percentage incidence of some of the skeletal parameters did attain statistical significance versus control, but these differences were not considered to be indicative of adverse foetal effects.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor the distribution of findings observed during detailed visceral examination indicated any adverse effect of maternal dietary exposure on foetal development.

At the low and intermediate dietary exposure levels (3000 and 7500 ppm), a statistically significant increase (p<0.05, p<0.01 respectively) in the foetal incidence of non-uniform patterning of the rugae was observed. The foetal incidence at both levels were within the historical control range and neither the foetal nor the litter incidence of this parameter in the 15000 ppm dietary exposure group differed significantly from the control. As there was no dose related response the higher incidence of this finding at 3000 and 7500 ppm compared with controls is not considered toxicologically significant in isolation.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
other: Developmental Toxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: Skeletal findings: reduced ossification; wavy rib; delays in skeletal development
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Table 2. Group Mean Body Weight Change Values

Dietary Concentration (ppm)

 

 

Cumulative Body Weight Change (g) from Day 3 of Gestation

4

5

8

11

14

17

20

 

0 (Control)

Mean

3.6

7.6

25.0

47.8

62.6

90.5

141.9

SD

8.7

7.1

8.0

9.3

11.4

18.5

20.2

n

24

24

24

24

24

24

24

 

 

3000

Mean

4.2

6.8

25.5

47.8

62.9

86.3

130.0

SD

6.7

6.8

8.5

10.3

10.9

13.0

18.2

n

24

24

24

24

24

24

24

 

 

7500

Mean

2.5

9.8

26.5

45.2

60.6

86.5

131.5

SD

8.8

7.6

11.1

12.0

13.6

17.1

21.0

n

24

24

24

24

24

24

24

 

 

15000

Mean

1.5

5.3

19.5

35.8**

51.4**

74.9**

116.3***

SD

9.5

11.7

15.5

18.7

18.8

20.0

19.2

n

24

24

24

24

24

24

24

Table 3. Group Mean Food Consumption Values

Dietary Concentration (ppm)

 

Food Consumption (g/rat/day)

between Days of Gestation

3-5

5-8

8-11

11-14

14-17

17-20

 

0 (Control)

Mean

18.0

21.8

24.8

23.7

26.8

27.0

SD

3.7

3.4

3.2

3.4

3.7

3.4

n

24

24

24

24

24

24

 

 

3000

Mean

17.1

21.7

24.2

24.9

25.6

26.0

SD

4.4

3.3

2.9

3.0

2.8

3.7

n

24

24

24

24

24

24

 

 

7500

Mean

18.7

21.3

23.3

24.4

26.8

26.9

SD

3.3

2.4

2.1

2.3

3.3

3.1

n

24

24

24

24

24

24

 

 

15000

Mean

15.1**

18.7**

20.1***

22.9

24.9

25.8

SD

1.9

3.4

2.8

2.9

4.7

2.2

n

24

24

24

24

24

24

Table 4. Group Mean Litter Data Values

Dietary Concentration (ppm)

 

Mean Male

Foetal Weight (g)

Mean Female

Foetal Weight (g)

Mean Foetal

Weight (g)

Mean

Placental

Weight (g)

Litter

Weight

(g)

Total

Placental

Weight

(g)

 

0 (Control)

Mean

4.173

3.961

4.067

0.576

54.353

7.675

SD

0.228

0.265

0.237

0.064

10.790

1.624

n

24

24

24

24

24

24

 

 

3000

Mean

3.986*

3.782

3.877*

0.553

49.742

7.110

SD

0.295

0.313

0.289

0.067

8.359

1.464

n

24

24

24

24

24

24

 

 

7500

Mean

3.956*

3.780

3.870*

0.571

49.805

7.233

SD

0.243

0.246

0.233

0.109

10.162

1.490

n

24

24

24

24

24

24

 

 

15000

Mean

3.636***

3.447***

3.545***

0.519

45.258***

6.658**

SD

0.260

0.228

0.245

0.063

5.559

1.249

n

24

24

24

24

24

24

Table 5. Summary Incidence of Foetal Skeletal Findings

 

 

Skeletal Findings

Dietary Concentration (ppm)

0 (Control)

3000

7500

15000

Number of Foetuses (litters) Examined

155 (24)

148 (24)

148 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

Frontal - incomplete ossification

7

4

4.7

4

4

3.0

14

7

9.3

31

14

20.1**

Frontal - unossified area

0

0

0.0

5

3

3.6

4

1

2.8

7

6

4.5*

Parietal - incomplete ossification

15

8

9.8

8

5

5.6

13

10

8.3

41

15

27.2*

Interparietal - incomplete ossification

31

13

20.1

29

12

20.4

35

12

23.3

74

22

49.8***

Occipital (Supra-occipital) - incomplete ossification

24

9

15.2

32

15

22.1

48

19

32.6**

73

21

48.9***

Squamosal - incomplete ossification

20

8

13.1

16

12

9.9

26

13

17.3

54

19

36.3**

Squamosal - unossified area(s)

8

6

5.7

1

1

0.7*

4

3

2.5

0

0

0.0*

Jugal - incomplete ossification

16

9

10.5

8

6

6.1

17

10

11.9

33

16

22.3*

Zygomatic process of maxilla - incomplete ossification

15

8

9.4

6

5

3.9

26

13

17.4

31

15

20.7*

Zygomatic process of squamosal - incomplete ossification

4

2

2.4

0

0

0.0

4

3

3.5

17

9

11.8*

Premaxilla - incomplete ossification

0

0

0.0

4

2

1.9

8

5

5.1

13

5

7.7*

Presphenoid - not ossified

1

1

0.5

1

1

0.5

0

0

0.0

11

7

6.9*

Vertebral Column

Lumbar (neural) arch - incomplete ossification

1

1

0.6

0

0

0.0

2

1

1.0

9

6

6.2*

Sacral (neural) arch - incomplete ossification

19

9

11.8

18

13

12.3

44

17

29.6

47

14

29.8*

Caudal vertebrae –

less than 4 ossified

18

12

11.5

39

16

25.5

38

14

24.8

60

18

38.3**

Ribs

Ossification centre - associated with 1st lumbar vertebra

13

9

9.1

3

3

1.8*

8

7

5.4

3

2

1.9*

One or more ribs – wavy

2

1

1.4

4

2

3.8

5

4

4.0

27

10

17.9**

One or more ribs – thickened

2

2

1.4

4

3

3.5

6

5

4.6

23

9

15.7*

Rib - incomplete ossification

3

2

2.0

1

1

1.0

3

2

1.9

20

8

13.4*

Sternebrae

Sternebra - not ossified

0

0

0.0

0

0

0.0

6

1

3.1

7

5

4.3*

Xiphoid cartilage - partially split

9

5

7.9

20

11

15.7

9

7

6.7

23

15

14.9*

Pelvic Girdle

Pubis - not ossified

0

0

0.0

0

0

0.0

3

3

1.8

10

5

6.4*

Pubis - incomplete ossification

11

6

6.5

6

6

4.1

26

12

16.6

31

11

20.6*

Forelimb

Metacarpal - not ossified 

18

10

12.1

40

18

27.4*

57

15

36.5*

81

20

52.7***

Metacarpal - incomplete ossification

3

3

2.3

1

1

0.8

10

6

6.7

22

11

14.4**

NF: Number of foetuses

NL: Number of litters

%†: Group mean percent

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Conclusions:
The oral administration of the test material to pregnant rats by continuous dietary admixture from gestation Days 3 to 19, at a dietary concentration of 15000 ppm (equivalent to a mean achieved dosage of 1164.7 mg/kg bw/day) was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. Therefore, the 7500 ppm dietary exposure level (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day) was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
 
In-utero survival of the developing conceptus was unaffected by maternal dietary exposure to 15000 ppm of the test item, although reduced foetal and placental weights and skeletal findings indicated an adverse effect on foetal growth. The NOAEL for developmental toxicity was therefore considered to be 7500 ppm (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day).
Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, fumarated, esters with glycerol based on category read across that is explained in the category justification document attached in Section 13 of the dossier.

In a key developmental toxicity study, the test material (Resin acids and Rosin acids, fumarated esters with Glycerol; CAS# 97489-11-7) was administered by continuous dietary admixture to three groups, each composed of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between gestation days 3 and 19 (inclusive) at dietary concentrations of 3000, 7500, or 15000 ppm (equivalent to mean achieved dosages of 244.2, 622.2 or 1164.7 mg/kg bw/day, respectively). A further group of twenty-four time mated females was fed basal laboratory diet to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were 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 weight, sex and external and internal macroscopic appearance were recorded. Half of the pups from each litter were examined for detailed skeletal development and the remainder were subjected to detailed visceral examination.

 

Dietary exposure to 15000 ppm of the test item was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. While part of the lower overall weight gain observed was attributable to lower litter weight due to reduced foetal weight, an underlying effect on the pregnant dam was still present when body weight gain was adjusted for the contribution of the gravid uterus. For females at the 3000 and 7500 ppm dietary exposure levels, clinical signs, body weight performance, food consumption and macroscopic necropsy examinations did not indicate any obvious effect of treatment.

 

In-utero survival of the developing conceptus appeared unaffected by maternal dietary exposure to 15000 ppm of the test item with both pre-and post-implantation losses being comparable to control. This was despite a clear treatment-related reduction in foetal weight which resulted in lower litter weight at this dietary exposure level and which attained statistical significance and was significantly different than the other dietary exposure groups.

 

At the 7500 and 3000 ppm dietary exposure levels there was a reduction in foetal weight compared with controls. However, this finding was considered not to be adverse because the mean foetal weights were within the historical control range (2.42 - 4.97g) and were within one standard deviation of the mean foetal weight of the control group. Furthermore, the difference in mean foetal weights between the low and intermediate dietary exposure groups was very marginal when compared with the difference in exposure levels, which is not suggestive of a clear indication of a relationship between maternal dietary exposure to 7500 and 3000 ppm of the test item and an effect on foetal weight.

 

Skeletal evaluation of foetuses from the 15000 ppm dietary exposure level showed significant differences compared to controls. The number of individual sites with reduced ossification and the difference in their incidence compared to controls was particularly higher, with a wide range of structures affected. Included within this were rib effects such as wavy rib. At the 7500 and 3000 ppm dietary exposure level, there was no increase in rib effects and the observed differences in ossification were limited to an increased incidence of incomplete ossification of one cranial bone (supra occipital) an no ossification of the metacarpals. The range of historical control incidence of foetuses with incomplete ossification of the supra occipital bone is 4.9%to 17.3% and the range of historical control incidence of foetuses with no ossification of the metacarpals is 5.3% to 22.2%, which is suggestive of high variability in the ossification of these structures at the end of gestation. Generalised delays have a common ‘finger print’, characterised by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation (e.g. supra occipital bone and metacarpals) and denotes generalized growth delays with subsequent catch-up postnatally (Carney and Kimmel, 2007). Consequently, these isolated intergroup differences, in the absence of concomitant reductions in ossification of the other associated structures should be regarded as non-conclusive evidence of a foetal effect. Therefore, based on the findings in this study there was a clear difference in the effects observed between the 15000 ppm exposure level and the 7500 and 3000 ppm dietary exposure groups. The former clearly demonstrated a reduction in foetal weights and a significantly increased incidence of delays in skeletal development. At the 7500 and 3000 ppm dietary exposure levels the observations were within historical control incidence, of limited toxicological significance and were not considered to be indicative of adverse foetal effects.

 

The oral administration of the test material to pregnant rats by continuous dietary admixture from gestation Days 3 to 19, at a dietary concentration of 15000 ppm (equivalent to a mean achieved dosage of 1164.7 mg/kg bw/day) was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. Therefore, the 7500 ppm dietary exposure level (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day) was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

 

In-utero survival of the developing conceptus was unaffected by maternal dietary exposure to 15000 ppm of the test item, although reduced foetal and placental weights and skeletal findings indicated an adverse effect on foetal growth. The NOAEL for developmental toxicity was therefore considered to be 7500 ppm (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
deviations had no adverse impact on the scientific purpose 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
Remarks:
Crl:CD®(SD) IGS BR strain
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited,Margate, Kent
- Age at study initiation: not specified
- Weight at study initiation: 171 to 275g
- Fasting period before study: not specified
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelvehours darkness

IN-LIFE DATES: From: 2015-08-15 To: 2015-09-03

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were preparedon four occasions during the course of the study and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart U200 mixer.
- Storage temperature of food: stored at room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the dietary admixtures was previously determined by Envigo Research Limited, Shardlow, UK, Analytical Services (Harlan Study Number 41302579). Representative samples were taken of three of the admixtures and analyzed for concentration of RARA, fumarated, esters with glycerol (CAS 97489-11-7) at Envigo Research Limited., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 85 % to 105 % of the nominal concentration confirming the suitability and accuracy of the formulation procedure.
Details on mating procedure:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited,Margate, Kent. Animals were delivered intwo batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
gestation days 3 to 19 (inclusive)
Frequency of treatment:
continuously in the diet
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
3 000 ppm
Remarks:
Low (equivalent to 244.2 mg/Kg bw/day)
Dose / conc.:
7 500 ppm
Remarks:
Intermediate (equivalent to 622.2 mg/Kg bw/day)
Dose / conc.:
15 000 ppm
Remarks:
High (equivalent to 1164.7 mg/Kg bw/day)
No. of animals per sex per dose:
24/concentration
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were selected in collaboration with the Sponsor Representative, based on available toxicity data including an OECD 422 Study in the rat (Harlan Study Number 41302579). In the OECD 422 Study, a dietary concentration of 15000 ppm was well tolerated during gestation, therefore dietary concentrations of 0 (Control), 3000, 7500 and 15000 ppm were selected for use in this pre-natal developmental study.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural change once daily. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on gestation days 3, 4, 5, 8, 11, 14, and 17. Body weights were also recorded for animals at terminal kill (Day 20).

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
Food consumption was recorded for each individual animal on gestation days 3, 5, 8, 11, 14, 17 and 20.

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 gestation day # 20
- Organs examined: All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on gestation day 20. All animals were subjected to a full external and internal examination. The ovaries and uteri of pregnant females were removed and examined.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
i) Foetal sex
ii) External foetal appearance
iii) Foetal weight
iv) Placental weight
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [all 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. All caesarean necropsy parameters and foetalparameters: 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. Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss
i) Percentage pre-implantation loss was calculated as: (Number of corpora lutea - Number of implantations/Number of corpora lutea) x 100
ii) Percentage post-implantation loss was calculated as: (Number of implantations - Number of live foetuses/Number of implantations) x 100

Sex Ratio
i) Sex ratio was calculated as: % male foetuses (sex ratio) = (Number of male foetuses/Total number of foetuses) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were detected.

One female in the 15000 ppm dietary exposure group had red staining around the ano-genital region on Day 11 of gestation. In isolation thiswas considered incidental and unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, body weight gain was generally lower than control throughout gestation, with differences frequently attaining statistical significance and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by dietary exposure to 3000 or 7500 ppm of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, food consumption was generally lower than control between gestation days 3 and 11, with differences attaining statistical significance. A slight recovery in food consumption was evident thereafter.

Food consumption during gestation was unaffected by dietary exposure to 3000 or 7500 ppm
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were detected during the macroscopic examination of the pregnant females at termination on gestation day 20.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Details on maternal toxic effects:
Maternal dietary exposure to 3000, 7500 or 15000 ppm of the test item resulted in no effects on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and postimplantation losses. At the 15000 ppm dietary exposure level, placental weights were statistically significantly reduced.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: effects on body weight and food consumption at the highest concentration tested.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, mean foetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance.

At the 3000 and 7500 ppm dietary exposure levels, mean male foetal weights and mean foetal weights (both sexes combined) were statistically significantly lower than control. All individual mean litter values were within historical background control ranges (mean male foetal weight: 3.24-5.03g and mean foetal weight: 2.42-4.97g) and in isolation at these levels were considered to reflect normal biological variation.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor the distribution of findings observed during external examination of the foetuses at necropsy on gestation day 20 indicated any adverse effect of maternal dietary exposure on foetal development.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm exposure level, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of foetuses/litters increased compared with control and differences frequently attaining statistical significance. These parameters included incomplete ossification of the frontal, parietal, interparietal, occipital, squamosal, jugal, zygomatic process of maxilla and squamosal, premaxilla bones of the skull, no ossification of the frontal and presphenoid bones of the skull, incomplete ossification of the lumbar (neural) arch, incomplete/no ossification of the sacral (neural) arch, less than 4 caudal vertebrae ossified, no ossification of the sternebra, partially split xiphoid cartilage,wavy/thickened/incomplete ossification of the ribs, and no ossification/incomplete ossification of the pubis, and metacarpals. There was also a lower incidence of foetuses showing no ossification of the squamosal bone of the skull and ossification centre associated with 1st lumbar vertebra.

At the 3000 and 7500 ppm dietary exposure levels, the incidence and type of skeletal findings observed did not indicate any adverse effect of maternal treatment. The percentage incidence of some of the skeletal parameters did attain statistical significance versus control, but these differences were not considered to be indicative of adverse foetal effects.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor the distribution of findings observed during detailed visceral examination indicated any adverse effect of maternal dietary exposure on foetal development.

At the low and intermediate dietary exposure levels (3000 and 7500 ppm), a statistically significant increase (p<0.05, p<0.01 respectively) in the foetal incidence of non-uniform patterning of the rugae was observed. The foetal incidence at both levels were within the historical control range and neither the foetal nor the litter incidence of this parameter in the 15000 ppm dietary exposure group differed significantly from the control. As there was no dose related response the higher incidence of this finding at 3000 and 7500 ppm compared with controls is not considered toxicologically significant in isolation.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
other: Developmental Toxicity

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: Skeletal findings: reduced ossification; wavy rib; delays in skeletal development

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 2. Group Mean Body Weight Change Values

Dietary Concentration (ppm)

 

 

Cumulative Body Weight Change (g) from Day 3 of Gestation

4

5

8

11

14

17

20

 

0 (Control)

Mean

3.6

7.6

25.0

47.8

62.6

90.5

141.9

SD

8.7

7.1

8.0

9.3

11.4

18.5

20.2

n

24

24

24

24

24

24

24

 

 

3000

Mean

4.2

6.8

25.5

47.8

62.9

86.3

130.0

SD

6.7

6.8

8.5

10.3

10.9

13.0

18.2

n

24

24

24

24

24

24

24

 

 

7500

Mean

2.5

9.8

26.5

45.2

60.6

86.5

131.5

SD

8.8

7.6

11.1

12.0

13.6

17.1

21.0

n

24

24

24

24

24

24

24

 

 

15000

Mean

1.5

5.3

19.5

35.8**

51.4**

74.9**

116.3***

SD

9.5

11.7

15.5

18.7

18.8

20.0

19.2

n

24

24

24

24

24

24

24

Table 3. Group Mean Food Consumption Values

Dietary Concentration (ppm)

 

Food Consumption (g/rat/day)

between Days of Gestation

3-5

5-8

8-11

11-14

14-17

17-20

 

0 (Control)

Mean

18.0

21.8

24.8

23.7

26.8

27.0

SD

3.7

3.4

3.2

3.4

3.7

3.4

n

24

24

24

24

24

24

 

 

3000

Mean

17.1

21.7

24.2

24.9

25.6

26.0

SD

4.4

3.3

2.9

3.0

2.8

3.7

n

24

24

24

24

24

24

 

 

7500

Mean

18.7

21.3

23.3

24.4

26.8

26.9

SD

3.3

2.4

2.1

2.3

3.3

3.1

n

24

24

24

24

24

24

 

 

15000

Mean

15.1**

18.7**

20.1***

22.9

24.9

25.8

SD

1.9

3.4

2.8

2.9

4.7

2.2

n

24

24

24

24

24

24

Table 4. Group Mean Litter Data Values

Dietary Concentration (ppm)

 

Mean Male

Foetal Weight (g)

Mean Female

Foetal Weight (g)

Mean Foetal

Weight (g)

Mean

Placental

Weight (g)

Litter

Weight

(g)

Total

Placental

Weight

(g)

 

0 (Control)

Mean

4.173

3.961

4.067

0.576

54.353

7.675

SD

0.228

0.265

0.237

0.064

10.790

1.624

n

24

24

24

24

24

24

 

 

3000

Mean

3.986*

3.782

3.877*

0.553

49.742

7.110

SD

0.295

0.313

0.289

0.067

8.359

1.464

n

24

24

24

24

24

24

 

 

7500

Mean

3.956*

3.780

3.870*

0.571

49.805

7.233

SD

0.243

0.246

0.233

0.109

10.162

1.490

n

24

24

24

24

24

24

 

 

15000

Mean

3.636***

3.447***

3.545***

0.519

45.258***

6.658**

SD

0.260

0.228

0.245

0.063

5.559

1.249

n

24

24

24

24

24

24

Table 5. Summary Incidence of Foetal Skeletal Findings

 

 

Skeletal Findings

Dietary Concentration (ppm)

0 (Control)

3000

7500

15000

Number of Foetuses (litters) Examined

155 (24)

148 (24)

148 (24)

148 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

Frontal - incomplete ossification

7

4

4.7

4

4

3.0

14

7

9.3

31

14

20.1**

Frontal - unossified area

0

0

0.0

5

3

3.6

4

1

2.8

7

6

4.5*

Parietal - incomplete ossification

15

8

9.8

8

5

5.6

13

10

8.3

41

15

27.2*

Interparietal - incomplete ossification

31

13

20.1

29

12

20.4

35

12

23.3

74

22

49.8***

Occipital (Supra-occipital) - incomplete ossification

24

9

15.2

32

15

22.1

48

19

32.6**

73

21

48.9***

Squamosal - incomplete ossification

20

8

13.1

16

12

9.9

26

13

17.3

54

19

36.3**

Squamosal - unossified area(s)

8

6

5.7

1

1

0.7*

4

3

2.5

0

0

0.0*

Jugal - incomplete ossification

16

9

10.5

8

6

6.1

17

10

11.9

33

16

22.3*

Zygomatic process of maxilla - incomplete ossification

15

8

9.4

6

5

3.9

26

13

17.4

31

15

20.7*

Zygomatic process of squamosal - incomplete ossification

4

2

2.4

0

0

0.0

4

3

3.5

17

9

11.8*

Premaxilla - incomplete ossification

0

0

0.0

4

2

1.9

8

5

5.1

13

5

7.7*

Presphenoid - not ossified

1

1

0.5

1

1

0.5

0

0

0.0

11

7

6.9*

Vertebral Column

Lumbar (neural) arch - incomplete ossification

1

1

0.6

0

0

0.0

2

1

1.0

9

6

6.2*

Sacral (neural) arch - incomplete ossification

19

9

11.8

18

13

12.3

44

17

29.6

47

14

29.8*

Caudal vertebrae –

less than 4 ossified

18

12

11.5

39

16

25.5

38

14

24.8

60

18

38.3**

Ribs

Ossification centre - associated with 1st lumbar vertebra

13

9

9.1

3

3

1.8*

8

7

5.4

3

2

1.9*

One or more ribs – wavy

2

1

1.4

4

2

3.8

5

4

4.0

27

10

17.9**

One or more ribs – thickened

2

2

1.4

4

3

3.5

6

5

4.6

23

9

15.7*

Rib - incomplete ossification

3

2

2.0

1

1

1.0

3

2

1.9

20

8

13.4*

Sternebrae

Sternebra - not ossified

0

0

0.0

0

0

0.0

6

1

3.1

7

5

4.3*

Xiphoid cartilage - partially split

9

5

7.9

20

11

15.7

9

7

6.7

23

15

14.9*

Pelvic Girdle

Pubis - not ossified

0

0

0.0

0

0

0.0

3

3

1.8

10

5

6.4*

Pubis - incomplete ossification

11

6

6.5

6

6

4.1

26

12

16.6

31

11

20.6*

Forelimb

Metacarpal - not ossified 

18

10

12.1

40

18

27.4*

57

15

36.5*

81

20

52.7***

Metacarpal - incomplete ossification

3

3

2.3

1

1

0.8

10

6

6.7

22

11

14.4**

NF: Number of foetuses

NL: Number of litters

%†: Group mean percent

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to pregnant rats by continuous dietary admixture from gestation Days 3 to 19, at a dietary concentration of 15000 ppm (equivalent to a mean achieved dosage of 1164.7 mg/kg bw/day) was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. Therefore, the 7500 ppm dietary exposure level (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day) was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.
 
In-utero survival of the developing conceptus was unaffected by maternal dietary exposure to 15000 ppm of the test item, although reduced foetal and placental weights and skeletal findings indicated an adverse effect on foetal growth. The NOAEL for developmental toxicity was therefore considered to be 7500 ppm (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day).
Executive summary:

In a key developmental toxicity study, the test material (Resin acids and Rosin acids, fumarated esters with Glycerol; CAS# 97489-11-7) was administered by continuous dietary admixture to three groups, each composed of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between gestation days 3 and 19 (inclusive) at dietary concentrations of 3000, 7500, or 15000 ppm (equivalent to mean achieved dosages of 244.2, 622.2 or 1164.7 mg/kg bw/day, respectively). A further group of twenty-four time mated females was fed basal laboratory diet to serve as a control.

 

Clinical signs, body weight change, food and water consumptions were 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 weight, sex and external and internal macroscopic appearance were recorded. Half of the pups from each litter were examined for detailed skeletal development and the remainder were subjected to detailed visceral examination.

 

Dietary exposure to 15000 ppm of the test item was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. While part of the lower overall weight gain observed was attributable to lower litter weight due to reduced foetal weight, an underlying effect on the pregnant dam was still present when body weight gain was adjusted for the contribution of the gravid uterus. For females at the 3000 and 7500 ppm dietary exposure levels, clinical signs, body weight performance, food consumption and macroscopic necropsy examinations did not indicate any obvious effect of treatment.

 

In-utero survival of the developing conceptus appeared unaffected by maternal dietary exposure to 15000 ppm of the test item with both pre-and post-implantation losses being comparable to control. This was despite a clear treatment-related reduction in foetal weight which resulted in lower litter weight at this dietary exposure level and which attained statistical significance and was significantly different than the other dietary exposure groups.

 

At the 7500 and 3000 ppm dietary exposure levels there was a reduction in foetal weight compared with controls. However, this finding was considered not to be adverse because the mean foetal weights were within the historical control range (2.42 - 4.97g) and were within one standard deviation of the mean foetal weight of the control group. Furthermore, the difference in mean foetal weights between the low and intermediate dietary exposure groups was very marginal when compared with the difference in exposure levels, which is not suggestive of a clear indication of a relationship between maternal dietary exposure to 7500 and 3000 ppm of the test item and an effect on foetal weight.

 

Skeletal evaluation of foetuses from the 15000 ppm dietary exposure level showed significant differences compared to controls. The number of individual sites with reduced ossification and the difference in their incidence compared to controls was particularly higher, with a wide range of structures affected. Included within this were rib effects such as wavy rib. At the 7500 and 3000 ppm dietary exposure level, there was no increase in rib effects and the observed differences in ossification were limited to an increased incidence of incomplete ossification of one cranial bone (supra occipital) an no ossification of the metacarpals. The range of historical control incidence of foetuses with incomplete ossification of the supra occipital bone is 4.9%to 17.3% and the range of historical control incidence of foetuses with no ossification of the metacarpals is 5.3% to 22.2%, which is suggestive of high variability in the ossification of these structures at the end of gestation. Generalised delays have a common ‘finger print’, characterised by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation (e.g. supra occipital bone and metacarpals) and denotes generalized growth delays with subsequent catch-up postnatally (Carney and Kimmel, 2007). Consequently, these isolated intergroup differences, in the absence of concomitant reductions in ossification of the other associated structures should be regarded as non-conclusive evidence of a foetal effect. Therefore, based on the findings in this study there was a clear difference in the effects observed between the 15000 ppm exposure level and the 7500 and 3000 ppm dietary exposure groups. The former clearly demonstrated a reduction in foetal weights and a significantly increased incidence of delays in skeletal development. At the 7500 and 3000 ppm dietary exposure levels the observations were within historical control incidence, of limited toxicological significance and were not considered to be indicative of adverse foetal effects.

 

The oral administration of the test material to pregnant rats by continuous dietary admixture from gestation Days 3 to 19, at a dietary concentration of 15000 ppm (equivalent to a mean achieved dosage of 1164.7 mg/kg bw/day) was associated with lower maternal body weight gain during gestation and an initial effect on food consumption. Therefore, the 7500 ppm dietary exposure level (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day) was considered to represent the No Observed Adverse Effect Level (NOAEL) for the pregnant female.

 

In-utero survival of the developing conceptus was unaffected by maternal dietary exposure to 15000 ppm of the test item, although reduced foetal and placental weights and skeletal findings indicated an adverse effect on foetal growth. The NOAEL for developmental toxicity was therefore considered to be 7500 ppm (equivalent to a mean achieved dosage of 622.2 mg/kg bw/day).