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:
2015-09-08 to 2016-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
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:
2015-09-08 to 2016-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs apparent for adult females throughout the study that were considered to indicate an effect of exposure tothe test item at dietary concentrations of 3000, 7500 or 15000 ppm.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For females in the 15000 ppm dietary concentration group, a statistically significant mean body weight loss was apparent during the first two days of dietary exposure. Although recovery of body weight gain was apparent for the remainder of gestation, body weight gain between gestation days 5 and 11 was statistically significantly lower than control. The initial body weight loss, followed by a period of lower body weight gain, resulted in statistically significant lower cumulative body weight gain from the start of treatment throughout gestation, relative to control. Body weight was also statistically significantly lower than control from gestation day 5. After adjustment for the contribution of the gravid uterus, final body weight and overall body weight gain still remained statistically significantly lower than control.

For females exposed to dietary concentration of 7500 ppm of the test item, a slight but statistically significant mean body weight losswas apparent during first day of dietary exposure. Cumulative body weight gain from the start of treatment was statistically significantly lower than control. However, when adjusted for the contribution of the gravid uterus, the lower overall body weight gain was not statistically different compared to control. Group mean body weight during gestation, including final body weight adjusted for the contribution of the gravid uterus, showed no statistically significant differences from control.

For females exposed to a dietary concentration of 3000 ppm of the test item, a slight but statistically significant mean body weight losswas apparent during first day of dietary exposure. Cumulative body weight gain from the start of treatment to gestation day 5 remained statistically significantly lower than control. Overall body weight gain, including when adjusted for the contribution of the gravid uterus, was similar to control and did not attain statistical significance. Group mean body weight during gestation, including final body weight adjusted for the contribution of the gravid uterus, showed no statistically significant differences from control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For females exposed to a dietary concentration of 15000 ppm of the test item, food consumption was statistically significantly lower than control throughout gestation, with differences from control being particularly marked during the first two days of dietary exposure.

For females exposed to a dietary concentration of 7500 ppm of the test item, food consumption was statistically significantly lower than control to gestation day 14, with differences being notably lower than control during the first two days of dietary exposure. Food consumption from gestation day 14 was similar to control for the remainder of gestation.

There was considered to be no effect of dietary exposure at 3000 ppm of the test item throughout gestation. Although lower food intake during gestation days 5 to 8 attained statistical significance when compared to control, this small isolated difference from controls was not considered to be adverse in view of the lack of effects on body weight or body weight gain.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did notreveal any overt intergroup differences at 3000, 7500 or 15000 ppm of the test item.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected for adult animals at necropsy on gestation day 20 at 3000, 7500 or 15000 ppm of the test item.
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
Other effects:
no effects observed
Description (incidence and severity):
Placental weights were considered to be similar to control and remained unaffected by treatment at the highest concentration tested.
Details on maternal toxic effects:
Maternal dietary exposure to 3000, 7500 or 15000 ppm of the test item produced 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 post-implantation losses.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, mean foetal and litter weights were statistically significantly lower than control. There was no effect of maternal dietary exposure to 3000 or 7500 ppm of the test item on mean foetal weights or placental weight. At 3000 ppm of the test item, statistically significantly higher male foetal weights were observed. This is not considered to be an adverse effect and, in the absence of any similar findings in females or at higher dietary exposure levels, was considered to be incidental and unrelated to maternal exposure to the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, the incidence of small foetuses was statistically significantly higher than control; this finding was consistent with lower foetal weights apparent at this dietary level. The incidence of other findings at this dietary exposure level did not indicate any obvious effect of maternaldietary exposure to the test item on foetal growth or development. At the 3000 or 7500 ppm dietary exposure level, neither the type, incidence nor the distribution of external foetal findings observed indicated any effect of maternal dietary exposure to the test item.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, there was a number of findings observed suggestive of a delay in ossification compared to controls, including:
- statistically significant increasesin the foetal incidence of unossified areas of the occipital bone, incomplete ossification of the thoracic centrum and less than four ossified pre-sacral vertebrae.
- statistically significant decreasesin the foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified.

However, in contrast, the foetal incidence of incomplete ossification of the parietal, intraparietal and jugal bones, the pubis and the femur was statistically significantly lower than the concurrent control incidence

Maternal dietary exposure to 3000 or 7500 ppm of the test item produced no effects on the type, incidence or distribution of skeletal findings observed in foetuses.

At the 7500 ppm dietary exposure level, there was a statistically significantly lower foetal incidence of unossified hyoid bones, compared to control, but in isolation and in the absence of any similar decrease at 15000 ppm,this finding was considered to be incidental and unrelated to maternal exposure to the test item.

At the 3000 ppm dietary exposure level, there was a statistically significantly lower foetal incidence of incomplete ossification of the interparietal and jugal bones compared to control. In view of the lack of dosage relationship these isolated findings were considered to be incidental and unrelated to maternal exposure to the test item.

At the 3000, 7500 or 15000 ppm dietary exposure level, there was a lower foetal incidence of incomplete ossification of the sacral arch, with differences at 3000 and 15000 ppm attaining statistical significance. However,
in the absence of any dosage relationship this finding was considered to be incidental and unrelated to maternal exposure to the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor the distribution of foetal visceral findings observed indicated any effect of maternal dietary exposure to the test item at 3000, 7500 or 15000 ppm.
Other effects:
no effects observed
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
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: unossified areas of occipital bone; incomplete ossification of thoracic centrum; less than four ossified pre-sacral vertebrae; decreased foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
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

2.8

8.0

23.2

43.3

57.3

89.2

137.7

SD

9.9

7.3

8.8

8.1

9.2

9.9

12.0

n

24

24

24

24

24

24

24

 

 

3000

Mean

-4.0**

3.5*

18.6

40.4

57.1

85.8

139.7

SD

6.3

5.1

7.2

8.9

9.6

10.8

14.3

n

24

24

24

24

24

24

24

 

 

7500

Mean

-5.4**

-1.9***

13.4**

31.9**

47.2**

79.3*

124.9*

SD

6.0

8.1

10.9

11.7

10.7

13.6

19.2

n

23

23

23

23

23

23

23

 

 

15000

Mean

-12.2***

-16.0***

-8.5***

5.8***

19.8***

52.1***

93.8***

SD

4.4

7.1

11.5

13.0

11.6

15.5

18.2

n

24

24

24

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

 

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

16.9

21.0

23.5

24.0

27.7

27.0

SD

3.9

4.4

4.7

3.1

2.3

2.7

n

24

24

24

24

24

24

 

 

3000

Mean

16.3

18.9**

22.9

24.2

26.8

26.6

SD

4.6

2.1

5.7

1.9

2.5

3.0

n

24

24

24

24

24

24

 

 

7500

Mean

10.3***

17.3***

20.7**

21.8*

26.4

26.2

SD

2.1

2.9

2.9

2.2

2.4

2.9

n

23

23

23

23

23

23

 

 

15000

Mean

6.6***

11.6***

15.8***

18.7***

22.7***

23.5***

SD

3.9

2.2

2.6

2.7

3.9

3.1

n

24

24

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Table 4. Group Mean Litter Data Values

Dietary

Concentration

(ppm)

 

Mean Male

Foetal

Weight (g)

Mean Female

Foetal

Weight (g)

Mean Foetal

Weight (g)

Litter Weight

(g)

 

0 (Control)

Mean

4.075

3.914

3.991

54.312

SD

0.303

0.272

0.268

6.492

n

24

24

24

24

 

 

3000

Mean

4.266*

4.067

4.158

56.697

SD

0.312

0.307

0.307

8.040

n

24

24

24

24

 

 

7500

Mean

4.013

3.830

3.919

50.897

SD

0.241

0.254

0.226

6.802

n

23

23

23

23

 

 

15000

Mean

3.566***

3.428***

3.498***

48.251**

SD

0.185

0.185

0.201

6.326

n

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Table 5. Summary Incidence of Foetal Skeletal Findings

 

 

Skeletal Findings

Dietary Concentration (ppm)

0 (Control)

3000

7500

15000

Number of Foetuses (litters) Examined

156 (24)

158 (24)

144 (23)

160 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

Parietal - incomplete ossification

17

10

11.5

5

5

3.0

6

5

4.1

1

1

0.7**

Interparietal - incomplete ossification

33

16

21.9

14

9

8.4*

17

11

11.5

10

6

5.9**

Occipital (Supra-occipital) - unossified area(s)

3

3

2.1

4

3

2.8

8

7

5.5

18

12

11.4**

Jugal - incomplete ossification

17

11

11.5

5

5

2.9*

5

4

3.4

4

2

2.2**

Hyoid – not ossified

21

12

13.1

12

6

6.5

4

3

2.7**

15

8

9.2

Vertebral Column

Ventral arch of vertebra 1 - ossification present

35

15

23.7

55

17

35.2

37

17

25.8

10

9

6.7*

Thoracic centrum - incomplete ossification

3

3

2.0

8

6

4.8

10

5

6.8

26

15

17.2***

Sacral (neural) arch - incomplete ossification

39

16

25.8

14

8

8.3**

19

12

13.0

21

10

13.1*

Caudal vertebrae - less than 4 ossified

34

13

21.9

23

12

13.6

63

18

42.7*

119

24

73.8***

Pelvic Girdle

Pubis - incomplete ossification

12

8

8.0

6

5

3.6

5

5

3.3

0

0

0.0**

Forelimb

Metacarpal - not ossified 

46

15

28.8

26

15

16.1

46

19

31.9

82

19

50.5*

Forepaw phalanges –

1 or more - ossified 

22

11

13.7

41

15

26.6

7

4

4.7

4

3

2.5*

Hindlimbs

Femur - incomplete ossification

9

7

6.1

2

2

1.3

3

2

2.1

0

0

0.0**

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:
Based on the results of this study, a dietary exposure level of 3000 ppm (equivalent to an approximate dosage of 237.3 mg/Kg bw/day) is considered to be a clear No Observed Adverse Effect Level (NOAEL) for the pregnant dam. Maternal dietary exposure to 7500 ppm (equivalent to an approximate dosage of 554.4 mg/kg bw/day) produced no obvious effects on in utero survival of the developing conceptus, foetal, litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control. Therefore a dietary level of 7500 ppm represents a clear NOAEL for the developing conceptus.
Executive summary:

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

In a key pre-natal developmental toxicity study, the test material (Resin acids and Rosin acids, methyl ester; CAS# 68186-14-1) was administered by continuous dietary admixture to three groups each 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, and 15000 ppm. A further group of twenty-four time mated females was treated with basal laboratory diet to serve as a control.

 

Clinical signs, body weight change, food and water consumption 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 each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

No mortality was observed through the study period. Maternal dietary exposure to 15000 ppm of the test material during gestation was associated with lower body weight gain and food consumption compared to control, with differences being particularly marked during the initial two days of dietary exposure. Although lower litter weight due to reduced foetal weight would have contributed to the lower final body weight and overall weight gain, an underlying effect on the pregnant dam was still clearly present when these values were adjusted for the contribution of the gravid uterus. Despite these marked effects on body weight and food intake, in utero survival of the developing conceptus appeared to be unaffected by maternal exposure to the test item at this dietary level although there was a clear reduction in foetal weight, which resulted in lower litter weight compared to control. The lower foetal weight at 15000 ppm of the test item is suggestive of a retardation of foetal growth and was supported by a slightly increased incidence of small foetuses compared to control.

 

Skeletal examination of the foetuses also revealed statistically significant differences from control for a number of skeletal parameters (increased foetal incidences of unossified areas of the occipital bone, incomplete ossification of the thoracic centrum and less than four ossified pre-sacral vertebrae and decreased foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified) consistent with such a retardation of growth. However, lower foetal incidence of incomplete ossification of the parietal, intraparietal and jugal bones, the pubis and the femur also attained statistical significance compared to control: this was considered contradictory to a delay in foetal maturation. Overall, the differences from control for foetal parameters showed a non-specific and inconsistent pattern of delayed ossification.

 

Maternal dietary exposure to 7500 ppm of the test material during gestation was generally well tolerated by the parent females. Although a slight mean body weight loss was apparent during the first day of treatment, this most probably reflects an initial reluctance to eat the diet as subsequent body weight gain was essentially similar to control. Food consumption was statistically significantly lower than control until gestation day 14, although differences were most notable during the first two days of dietary exposure. There was no obvious effect of maternal exposure to the test item at a concentration of 7500 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control.

 

Maternal dietary exposure to 3000 ppm of the test material during gestation was well tolerated by the parent females. Although a slight mean body weight loss was apparent during the first day of treatment, this was considered to be of no toxicological significance in the absence of any effect on overall body weight gain during gestation compared to control. There was no obvious effect of maternal exposure to the test item at a concentration of 3000 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control.

 

At 7500 ppm, food consumption was lower than control throughout much of gestation and this lower food intake was associated with lower cumulative body weight gains during gestation. The clear NOAEL was considered to be 3000 ppm (equivalent to 237.3 mg/kg bw/day). There was no obvious effect of maternal exposure to the test item at a concentration of 7500 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control. Therefore, 7500 ppm (equivalent to 554.4 mg/kg bw/day) was considered to be the NOAEL for the survival, growth and development of the offspring.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Deviation was considered to have had no impact on the purpose or validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Strain: Crl:CD®(SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited,Margate, Kent.
- Age at study initiation: Animals were delivered in two batches containing females prior to Day 3 of gestation
- Weight at study initiation: 193 to 299g
- Fasting period before study: Not specified
- Housing: 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 (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) was provided 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: 2015-09-11 To: 2015-09-30

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 prepared in one batch and stored at room temperature.
- Mixing appropriate amounts with (Type of food): A known amount of Rosin, methyl ester CAS 68186-14-1 was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer4 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 H800 mixer.
- Storage temperature of food: stable for 24 days at room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dietary admixtures were prepared in one batch and stored at room temperature. Representative samples were taken from the dietary admixtures and analyzed for concentration and homogeneity of Rosin, methyl ester CAS 68186-14-1 by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 97 % to 100 % 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 in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
The test item was administered continuously in the diet, from gestation day 3 to 19
Frequency of treatment:
continuously in the diet
Duration of test:
Gestation day 3 to 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
3 000 ppm
Remarks:
Low (Mean Achieved Dosage = 237.3 mg/Kg bw/day)
Dose / conc.:
7 500 ppm
Remarks:
Intermediate (Mean Achieved Dosage = 554.4 mg/Kg bw/day)
Dose / conc.:
15 000 ppm
Remarks:
High (Mean Achieved Dosage = 962.5 mg/Kg bw/day)
No. of animals per sex per dose:
24 females/concentration
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dietary concentrations were selected in collaboration with the sponsor based on available toxicity data, including a preliminary pre-natal study in the rat performed at this Test facility (Study Number: 41403697). In the preliminary study, there were no findings observed that precluded the use of 15000 ppm as the highest dietary concentration in this pre-natal development toxicity study, therefore dietary concentrations of 0 (Control), 3000, 7500 and 15000 ppm were selected.
- 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: Once daily

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each individual animal on gestation day 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 toa full external and internal examination and any macroscopic abnormalities were recorded. 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
ivi) 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
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, on 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:
1) Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as: (Number of Corpora lutea - Number of Implantations/Number of Corpora lutea) x 100

Percentage post-implantation loss was calculated as: (Number of Implantations - Number of Live foetuses/Number of Implantations) x 100

2) Sex Ratio
Sex ratio was calculated as: % male foetuses (sex ratio) = ((Number of Male foetuses/Total number of foetuses) x 100
Historical control data:
No

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs apparent for adult females throughout the study that were considered to indicate an effect of exposure tothe test item at dietary concentrations of 3000, 7500 or 15000 ppm.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For females in the 15000 ppm dietary concentration group, a statistically significant mean body weight loss was apparent during the first two days of dietary exposure. Although recovery of body weight gain was apparent for the remainder of gestation, body weight gain between gestation days 5 and 11 was statistically significantly lower than control. The initial body weight loss, followed by a period of lower body weight gain, resulted in statistically significant lower cumulative body weight gain from the start of treatment throughout gestation, relative to control. Body weight was also statistically significantly lower than control from gestation day 5. After adjustment for the contribution of the gravid uterus, final body weight and overall body weight gain still remained statistically significantly lower than control.

For females exposed to dietary concentration of 7500 ppm of the test item, a slight but statistically significant mean body weight losswas apparent during first day of dietary exposure. Cumulative body weight gain from the start of treatment was statistically significantly lower than control. However, when adjusted for the contribution of the gravid uterus, the lower overall body weight gain was not statistically different compared to control. Group mean body weight during gestation, including final body weight adjusted for the contribution of the gravid uterus, showed no statistically significant differences from control.

For females exposed to a dietary concentration of 3000 ppm of the test item, a slight but statistically significant mean body weight losswas apparent during first day of dietary exposure. Cumulative body weight gain from the start of treatment to gestation day 5 remained statistically significantly lower than control. Overall body weight gain, including when adjusted for the contribution of the gravid uterus, was similar to control and did not attain statistical significance. Group mean body weight during gestation, including final body weight adjusted for the contribution of the gravid uterus, showed no statistically significant differences from control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For females exposed to a dietary concentration of 15000 ppm of the test item, food consumption was statistically significantly lower than control throughout gestation, with differences from control being particularly marked during the first two days of dietary exposure.

For females exposed to a dietary concentration of 7500 ppm of the test item, food consumption was statistically significantly lower than control to gestation day 14, with differences being notably lower than control during the first two days of dietary exposure. Food consumption from gestation day 14 was similar to control for the remainder of gestation.

There was considered to be no effect of dietary exposure at 3000 ppm of the test item throughout gestation. Although lower food intake during gestation days 5 to 8 attained statistical significance when compared to control, this small isolated difference from controls was not considered to be adverse in view of the lack of effects on body weight or body weight gain.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did notreveal any overt intergroup differences at 3000, 7500 or 15000 ppm of the test item.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected for adult animals at necropsy on gestation day 20 at 3000, 7500 or 15000 ppm of the test item.

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
Other effects:
no effects observed
Description (incidence and severity):
Placental weights were considered to be similar to control and remained unaffected by treatment at the highest concentration tested.
Details on maternal toxic effects:
Maternal dietary exposure to 3000, 7500 or 15000 ppm of the test item produced 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 post-implantation losses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
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):
At the 15000 ppm dietary exposure level, mean foetal and litter weights were statistically significantly lower than control. There was no effect of maternal dietary exposure to 3000 or 7500 ppm of the test item on mean foetal weights or placental weight. At 3000 ppm of the test item, statistically significantly higher male foetal weights were observed. This is not considered to be an adverse effect and, in the absence of any similar findings in females or at higher dietary exposure levels, was considered to be incidental and unrelated to maternal exposure to the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, the incidence of small foetuses was statistically significantly higher than control; this finding was consistent with lower foetal weights apparent at this dietary level. The incidence of other findings at this dietary exposure level did not indicate any obvious effect of maternaldietary exposure to the test item on foetal growth or development. At the 3000 or 7500 ppm dietary exposure level, neither the type, incidence nor the distribution of external foetal findings observed indicated any effect of maternal dietary exposure to the test item.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At the 15000 ppm dietary exposure level, there was a number of findings observed suggestive of a delay in ossification compared to controls, including:
- statistically significant increasesin the foetal incidence of unossified areas of the occipital bone, incomplete ossification of the thoracic centrum and less than four ossified pre-sacral vertebrae.
- statistically significant decreasesin the foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified.

However, in contrast, the foetal incidence of incomplete ossification of the parietal, intraparietal and jugal bones, the pubis and the femur was statistically significantly lower than the concurrent control incidence

Maternal dietary exposure to 3000 or 7500 ppm of the test item produced no effects on the type, incidence or distribution of skeletal findings observed in foetuses.

At the 7500 ppm dietary exposure level, there was a statistically significantly lower foetal incidence of unossified hyoid bones, compared to control, but in isolation and in the absence of any similar decrease at 15000 ppm,this finding was considered to be incidental and unrelated to maternal exposure to the test item.

At the 3000 ppm dietary exposure level, there was a statistically significantly lower foetal incidence of incomplete ossification of the interparietal and jugal bones compared to control. In view of the lack of dosage relationship these isolated findings were considered to be incidental and unrelated to maternal exposure to the test item.

At the 3000, 7500 or 15000 ppm dietary exposure level, there was a lower foetal incidence of incomplete ossification of the sacral arch, with differences at 3000 and 15000 ppm attaining statistical significance. However,
in the absence of any dosage relationship this finding was considered to be incidental and unrelated to maternal exposure to the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor the distribution of foetal visceral findings observed indicated any effect of maternal dietary exposure to the test item at 3000, 7500 or 15000 ppm.
Other effects:
no effects observed

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

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: unossified areas of occipital bone; incomplete ossification of thoracic centrum; less than four ossified pre-sacral vertebrae; decreased foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
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

2.8

8.0

23.2

43.3

57.3

89.2

137.7

SD

9.9

7.3

8.8

8.1

9.2

9.9

12.0

n

24

24

24

24

24

24

24

 

 

3000

Mean

-4.0**

3.5*

18.6

40.4

57.1

85.8

139.7

SD

6.3

5.1

7.2

8.9

9.6

10.8

14.3

n

24

24

24

24

24

24

24

 

 

7500

Mean

-5.4**

-1.9***

13.4**

31.9**

47.2**

79.3*

124.9*

SD

6.0

8.1

10.9

11.7

10.7

13.6

19.2

n

23

23

23

23

23

23

23

 

 

15000

Mean

-12.2***

-16.0***

-8.5***

5.8***

19.8***

52.1***

93.8***

SD

4.4

7.1

11.5

13.0

11.6

15.5

18.2

n

24

24

24

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

 

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

16.9

21.0

23.5

24.0

27.7

27.0

SD

3.9

4.4

4.7

3.1

2.3

2.7

n

24

24

24

24

24

24

 

 

3000

Mean

16.3

18.9**

22.9

24.2

26.8

26.6

SD

4.6

2.1

5.7

1.9

2.5

3.0

n

24

24

24

24

24

24

 

 

7500

Mean

10.3***

17.3***

20.7**

21.8*

26.4

26.2

SD

2.1

2.9

2.9

2.2

2.4

2.9

n

23

23

23

23

23

23

 

 

15000

Mean

6.6***

11.6***

15.8***

18.7***

22.7***

23.5***

SD

3.9

2.2

2.6

2.7

3.9

3.1

n

24

24

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Table 4. Group Mean Litter Data Values

Dietary

Concentration

(ppm)

 

Mean Male

Foetal

Weight (g)

Mean Female

Foetal

Weight (g)

Mean Foetal

Weight (g)

Litter Weight

(g)

 

0 (Control)

Mean

4.075

3.914

3.991

54.312

SD

0.303

0.272

0.268

6.492

n

24

24

24

24

 

 

3000

Mean

4.266*

4.067

4.158

56.697

SD

0.312

0.307

0.307

8.040

n

24

24

24

24

 

 

7500

Mean

4.013

3.830

3.919

50.897

SD

0.241

0.254

0.226

6.802

n

23

23

23

23

 

 

15000

Mean

3.566***

3.428***

3.498***

48.251**

SD

0.185

0.185

0.201

6.326

n

24

24

24

24

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Table 5. Summary Incidence of Foetal Skeletal Findings

 

 

Skeletal Findings

Dietary Concentration (ppm)

0 (Control)

3000

7500

15000

Number of Foetuses (litters) Examined

156 (24)

158 (24)

144 (23)

160 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Skull

Parietal - incomplete ossification

17

10

11.5

5

5

3.0

6

5

4.1

1

1

0.7**

Interparietal - incomplete ossification

33

16

21.9

14

9

8.4*

17

11

11.5

10

6

5.9**

Occipital (Supra-occipital) - unossified area(s)

3

3

2.1

4

3

2.8

8

7

5.5

18

12

11.4**

Jugal - incomplete ossification

17

11

11.5

5

5

2.9*

5

4

3.4

4

2

2.2**

Hyoid – not ossified

21

12

13.1

12

6

6.5

4

3

2.7**

15

8

9.2

Vertebral Column

Ventral arch of vertebra 1 - ossification present

35

15

23.7

55

17

35.2

37

17

25.8

10

9

6.7*

Thoracic centrum - incomplete ossification

3

3

2.0

8

6

4.8

10

5

6.8

26

15

17.2***

Sacral (neural) arch - incomplete ossification

39

16

25.8

14

8

8.3**

19

12

13.0

21

10

13.1*

Caudal vertebrae - less than 4 ossified

34

13

21.9

23

12

13.6

63

18

42.7*

119

24

73.8***

Pelvic Girdle

Pubis - incomplete ossification

12

8

8.0

6

5

3.6

5

5

3.3

0

0

0.0**

Forelimb

Metacarpal - not ossified 

46

15

28.8

26

15

16.1

46

19

31.9

82

19

50.5*

Forepaw phalanges –

1 or more - ossified 

22

11

13.7

41

15

26.6

7

4

4.7

4

3

2.5*

Hindlimbs

Femur - incomplete ossification

9

7

6.1

2

2

1.3

3

2

2.1

0

0

0.0**

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:
Based on the results of this study, a dietary exposure level of 3000 ppm (equivalent to an approximate dosage of 237.3 mg/Kg bw/day) is considered to be a clear No Observed Adverse Effect Level (NOAEL) for the pregnant dam. Maternal dietary exposure to 7500 ppm (equivalent to an approximate dosage of 554.4 mg/kg bw/day) produced no obvious effects on in utero survival of the developing conceptus, foetal, litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control. Therefore a dietary level of 7500 ppm represents a clear NOAEL for the developing conceptus.
Executive summary:

In a key pre-natal developmental toxicity study, the test material (Resin acids and Rosin acids, methyl ester; CAS# 68186-14-1) was administered by continuous dietary admixture to three groups each 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, and 15000 ppm. A further group of twenty-four time mated females was treated with basal laboratory diet to serve as a control.

 

Clinical signs, body weight change, food and water consumption 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 each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

 

No mortality was observed through the study period. Maternal dietary exposure to 15000 ppm of the test material during gestation was associated with lower body weight gain and food consumption compared to control, with differences being particularly marked during the initial two days of dietary exposure. Although lower litter weight due to reduced foetal weight would have contributed to the lower final body weight and overall weight gain, an underlying effect on the pregnant dam was still clearly present when these values were adjusted for the contribution of the gravid uterus. Despite these marked effects on body weight and food intake, in utero survival of the developing conceptus appeared to be unaffected by maternal exposure to the test item at this dietary level although there was a clear reduction in foetal weight, which resulted in lower litter weight compared to control. The lower foetal weight at 15000 ppm of the test item is suggestive of a retardation of foetal growth and was supported by a slightly increased incidence of small foetuses compared to control.

 

Skeletal examination of the foetuses also revealed statistically significant differences from control for a number of skeletal parameters (increased foetal incidences of unossified areas of the occipital bone, incomplete ossification of the thoracic centrum and less than four ossified pre-sacral vertebrae and decreased foetal incidence of ossification present in ventral arch of vertebra 1 and one or more forepaw phalanges ossified) consistent with such a retardation of growth. However, lower foetal incidence of incomplete ossification of the parietal, intraparietal and jugal bones, the pubis and the femur also attained statistical significance compared to control: this was considered contradictory to a delay in foetal maturation. Overall, the differences from control for foetal parameters showed a non-specific and inconsistent pattern of delayed ossification.

 

Maternal dietary exposure to 7500 ppm of the test material during gestation was generally well tolerated by the parent females. Although a slight mean body weight loss was apparent during the first day of treatment, this most probably reflects an initial reluctance to eat the diet as subsequent body weight gain was essentially similar to control. Food consumption was statistically significantly lower than control until gestation day 14, although differences were most notable during the first two days of dietary exposure. There was no obvious effect of maternal exposure to the test item at a concentration of 7500 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control.

 

Maternal dietary exposure to 3000 ppm of the test material during gestation was well tolerated by the parent females. Although a slight mean body weight loss was apparent during the first day of treatment, this was considered to be of no toxicological significance in the absence of any effect on overall body weight gain during gestation compared to control. There was no obvious effect of maternal exposure to the test item at a concentration of 3000 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control.

 

At 7500 ppm, food consumption was lower than control throughout much of gestation and this lower food intake was associated with lower cumulative body weight gains during gestation. The clear NOAEL was considered to be 3000 ppm (equivalent to 237.3 mg/kg bw/day). There was no obvious effect of maternal exposure to the test item at a concentration of 7500 ppm on in utero survival of the developing conceptus, foetal litter or placental weights or the foetal incidence of external, visceral or skeletal findings compared to control. Therefore, 7500 ppm (equivalent to 554.4 mg/kg bw/day) was considered to be the NOAEL for the survival, growth and development of the offspring.