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:
2015-03-11 to 2016-05-03
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:
2015-03-11 to 2016-05-03
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):
No clinical signs were apparent in any treatment group during the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 18750 ppm, a mean body weight loss was apparent on the first day of dietary exposure, thereafter body weight gain was generally similar or slightly superior to control and cumulative body weight gain was essentially similar to control by gestation day 14. However, lower body weight gain was subsequently observed between gestation day 14 to 20, with differences from control attaining statistical significance between gestation days 14 and 17. Overall body weight remained lower than control, although differences were not statistically significant, when adjusted for the contribution of the gravid uterus.

Dietary exposure at 8750 and 3250 ppm produced no obvious effect on body weight gain during pregnancy, with or without adjustment for the contribution of the gravid uterus.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 18750 ppm, food consumption was lower than control during the first two days of dietary exposure (Days 3-5) probably reflecting an initial reluctance to eat the treated diet due to its palatability. Thereafter, food intake during pregnancy was considered to be similar to control. There was no obvious effect on food intake during pregnancy at 3250 and 8750 ppm. At 8750 ppm, higher food intake attained statistical significance when compared with control but, in isolation, this finding was considered incidental and unrelated to treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was unaffected by dietary exposure at 3250, 8750 and 18750 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were apparent for females during the study.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.
Details on maternal toxic effects:
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
8 750 ppm
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 18750 ppm, mean foetal weights and litter weight were lower than control, with differences from control attaining statistical significance. Mean placental and total placenta weights were also slightly lower than control but only values for total placental weight attained statistical significance.

At 8750 ppm, mean litter and placental weights wereconsidered to be similar to control. Mean foetal weights (combined and female) were statistically lower than control.

At 3250 ppm, mean foetal, litter and placental weight were similar to control.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 18750 ppm, mean foetal weights and litter weight were lower than control, with differences from control attaining statistical significance. Mean placental and total placenta weights were also slightly lower than control but only values for total placental weight attained statistical significance.

At 8750 ppm, mean litter and placental weights wereconsidered to be similar to control. Mean foetal weights (combined and female) were statistically lower than control.

At 3250 ppm, mean foetal, litter and placental weight were similar to control.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during external examination of the foetuses at necropsy on gestation day 20 indicated any obvious effect of maternal treatment on foetal development at 3250, 8750 or 18750 ppm.

The incidence of foetuses with gastroschisis and encephalocoele at 18750 ppm, often also with spina bifida, was higher than control and was also above the historical control incidence. Although these findings were limited to one litter, an association with treatment cannot be discounted.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during skeletal examination of the foetuses indicated any obvious effect of maternal treatment on foetal development.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral examination of foetuses at 18750 ppm, revealed an apparent disturbance of the development of the kidney and ureter. This was principally manifested as increased incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla compared with control, although a low incidence of misshapen kidneys and absent renal medulla was also observed at this dietary level. The incidences of these findings were higher than observed for the historical control range.

At 8750 ppm, the incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla was also higher than control and the historical control range.

At 3250 ppm, only the incidence of foetuses/litters showing increased renal pelvic cavitation was higher than both concurrent control and the historical control range. The incidences of foetuses/litters showing kinked ureters or dilated ureters were similar to control and were within the historical control range.

The type, incidence and distribution of other visceral findings did not indicate any other effect of maternal treatment on foetal development at 3250, 8750 or 18750 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
3 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: urinary
Description (incidence and severity):
Cluster of visceral findings (kinked ureters, dilated ureters, increased renal pelvic cavitation, absent renal papilla misshapen kidneys and absent renal medulla) observed at the 8750 and 18750 ppm concentration levels.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
8 750 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 5 of Gestation

4

5

8

11

14

17

20

 

0 (Control)

Mean

5.2

9.4

19.3

40.6

54.3

86.5

137.6

SD

7.5

7.6

8.8

10.3

10.8

13.7

20.4

n

23

23

23

23

23

23

23

 

 

3250

Mean

4.6

8.4

18.4

41.1

55.0

85.7

134.3

SD

7.4

6.3

8.3

9.6

10.5

12.8

17.3

n

23

23

23

23

23

23

23

 

 

8750

Mean

2.5

8.8

20.8

43.0

57.8

87.2

135.5

SD

6.9

7.5

9.9

10.1

11.2

14.7

21.3

n

24

24

24

24

24

24

24

 

 

18750

Mean

-3.9***

1.6**

12.0*

35.7

52.3

78.1

122.9

SD

6.9

8.0

10.0

8.8

11.8

13.9

18.6

n

23

23

23

23

23

23

23

* 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

21.4

20.8

23.1

23.9

26.0

26.1

SD

4.9

2.6

2.2

3.3

2.7

3.4

n

23

23

23

23

23

23

 

 

3250

Mean

20.9

21.2

24.0

25.0

25.3

26.1

SD

3.1

3.0

2.7

2.1

2.0

2.3

n

23

23

23

23

23

23

 

 

8750

Mean

20.6

22.1

23.8

26.3*

26.3

26.2

SD

4.8

2.5

4.2

2.8

2.2

3.4

n

24

24

24

24

24

24

 

 

18750

Mean

17.5*

19.0

22.9

25.6

25.4

25.2

SD

2.6

2.9

2.8

3.1

2.8

2.3

n

23

23

23

23

23

23

* 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)

Total Placental Weight

(g)

 

0 (Control)

Mean

4.091

3.906

3.994

7.576

SD

0.218

0.199

0.200

1.317

n

23

23

23

23

 

 

3250

Mean

3.949

3.766

3.861

8.133

SD

0.299

0.304

0.314

1.493

n

23

23

23

23

 

 

8750

Mean

3.960

3.781*

3.865*

7.418

SD

0.275

0.260

0.263

1.409

n

24

24

24

24

 

 

18750

Mean

3.765**

(3.849**)

3.596**

(3.683*)

3.671**

(3.755**)

6.894*

(7.135*)

SD

0.461

(0.233)

0.495

(0.268)

0.472

(0.251)

1.715

(1.298)

n

23

(22)

23

(22)

23

(22)

23

(22)

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

( ) = Excludes one litter with atypically low foetal weights and significant foetal abnormalities.

Table 5. Summary Incidence of Foetal Visceral Findings

 

 

Visceral Findings

Dietary Concentration (ppm)

0 (Control)

3250

8750

18750

Number of Foetuses (litters) Examined

157 (23)

165 (23)

169 (24)

151 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Abdomen

Ureter – kinked

24

14

15.2

27

10

17.4

50

19

32.0*

60

19

38.6**

Ureter - dilated - Slight/Severe

18

10

11.3

16

9

10.8

36

16

24.1

48

16

31.1*

Renal pelvic cavitation - increased -

Slight/Severe

10

8

6.3

24

13

14.1

26

11

18.4

47

18

30.0***

Renal papilla – absent

1

1

0.9

0

0

0.0

6

4

5.9

13

6

9.0*

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, the No Observed Adverse Effect Level (NOAEL) for the pregnant rat was considered to be 8750 ppm (equivalent to 715.0 mg/kg bw/day) and the NOAEL for the developing conceptus was considered to be 3250 ppm (equivalent to 266.7 mg/kg bw/day).
Executive summary:

This data is being read across from the source study that tested Resin acids and Rosin acids, esters with ethylene glycol 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 (Rosin, esters with glycerol; CAS# 68512-65-2), 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 3250, 8750, and 18750 ppm (equivalent to a mean achieved dosage of 266.7, 715.0 and 1487.0 mg/kg bw/day respectively). A further group of twenty-four time mated females was treated with basal laboratory diet to serve as a control. test material (Rosin, esters with glycerol; CAS# 68512-65-2), 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 3250, 8750, and 18750 ppm (equivalent to a mean achieved dosage of 266.7, 715.0 and 1487.0 mg/kg bw/day respectively). 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 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.

 

No mortality or signs of clinical toxicity were observed through the study period. Initial dietary exposure at 18750 ppm was associated with a mean body weight loss, thereafter body weight gain was similar to control to gestation day 14. Lower body weight gain was observed from gestation days 14 to 20. During the later stage of gestation, body weight gain in the high exposure group may have been influenced by lower foetal/litter weight, however overall body weight remained lower than control, when adjusted for the contribution of the gravid uterus. Food consumption at this dietary level was lower than control during the first two days of dietary exposure, which probably reflected an initial reluctance to eat the treated diet (due to its palatability), but subsequent food intake was similar to control.

 

For litters at 18750 ppm, there was no effect of maternal exposure on pre-implantation loss, implantation count, embryofoetal survival, litter size or sex ratio but mean foetal, litter and placental weight were all lower than control. External examination of the foetuses at necropsy and subsequent detailed skeletal evaluation did not indicate any effect of maternal exposure on foetal development. However, there was a cluster of visceral findings (kinked ureters, dilated ureters, increased renal pelvic cavitation, absent renal papilla misshapen kidneys and absent renal medulla) that indicated a treatment-related disturbance of the normal development of the kidneys and ureters. One litter at 18750 ppm showed five foetuses with gastroschisis and encephalocoele. Despite the limitation of this finding to one litter, the group mean incidence of affected foetuses was higher than both concurrent and historical control values. As this finding was observed in the highest treated group an association with treatment cannot be discounted resulting in confirmation that dietary exposure at 18750 ppm is regarded as an adverse effect level for offspring development.

 

At 3250 and 8750 ppm, there was no effect of dietary exposure on maternal body weight gain or food consumption and pre-implantation loss, implantation count, embryofoetal survival, litter size, sex ratio and mean foetal, litter and placental weight were also considered to be unaffected by maternal exposure at these dietary levels. However, at 8750 ppm, the incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla was higher than control and the historical control range, indicating a treatment-related effect on the normal development of the kidneys and ureters. At 3250 ppm, the incidences of foetuses/litters showing kinked ureters or dilated ureters were similar to control and were within the historical control range. However, the incidence of foetuses/litters showing increased renal pelvic cavitation was higher than both concurrent control and the historical control range. While this higher incidence of increased renal pelvic cavitation was only slightly higher than the historical control range, in view of the findings apparent at higher dietary levels, an association with treatment cannot be discounted. However, increased renal pelvic cavitation is frequently observed amongst neonatal and adult animals within this laboratory without any obvious adverse impact of the viability of these animals and may be regarded, to a certain extent, as a variation of the normal. As this finding occurred in the absence of any increased incidence of more significant findings with the potential to effect offspring viability, such as absent renal papilla, and in the absence of any effect on skeletal development, this dietary level could be regarded as a No Observed Adverse Effect Level (NOAEL) for foetal development.

 

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for the pregnant rat was considered to be 8750 ppm (equivalent to 715 mg/kg bw/day) and the NOAEL for developmental toxicity was considered to be 3250 ppm (equivalent to 266.7 mg/kg bw/day).

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:
Deviations are not considered to have had an adverse impact on the scientific integrity 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
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: 178 to 274 g
- 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 diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) was used 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 twelve hours of darkness

IN-LIFE DATES: From: 2015-03-13 To: 2015-04-01

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 once and stored at room temperature.
- Mixing appropriate amounts with (Type of food): A known amount of Rosin, ethylene glycol ester CAS 68512-65-2 was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous. This premix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 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 performed by the Test Facility during a Rat Dietary OECD 422 Toxicity Study and reported as Envigo Research Limited, Study Number 41302558. Results showed the dietary admixtures to be stable for at
least 27 days at room temperature.Samples were taken of each test item formulation and were analyzed for concentration of Rosin, ethylene glycol ester CAS 68512-65-2 at Harlan Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 125% of the nominal concentration.
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 prior to gestation day 3. The day that positive evidence of mating was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
The test material was administered continuously from Gestation day 3 to 19 by dietary admixture.
Frequency of treatment:
Administered continuously by dietary admixture.
Duration of test:
From Gestation day 3 to 19
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control
Dose / conc.:
3 250 ppm
Remarks:
Low Concentration (equivalent to a Mean Achieved Dose Level of 266.7 mg/Kg bw/day)
Dose / conc.:
8 750 ppm
Remarks:
Intermediate Concentration (equivalent to a Mean Achieved Dose Level of 715.0 mg/Kg bw/day)
Dose / conc.:
18 750 ppm
Remarks:
High Concentration (equivalent to a Mean Achieved Dose Level of 1487.0 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: The dietary concentrations were chosen based on previous toxicity data including a Rat Dietary OECD 422 Toxicity Study (Envigo Research Limited, Study Number 41302558). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the potential hazards of the test item to man.
- 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 once daily for overt signs of toxicity, ill-health or behavioural changes throughout the gestation period. 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 (gestation day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each individual animal at 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 to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
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:
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 foetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen. 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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were apparent in any treatment group during the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 18750 ppm, a mean body weight loss was apparent on the first day of dietary exposure, thereafter body weight gain was generally similar or slightly superior to control and cumulative body weight gain was essentially similar to control by gestation day 14. However, lower body weight gain was subsequently observed between gestation day 14 to 20, with differences from control attaining statistical significance between gestation days 14 and 17. Overall body weight remained lower than control, although differences were not statistically significant, when adjusted for the contribution of the gravid uterus.

Dietary exposure at 8750 and 3250 ppm produced no obvious effect on body weight gain during pregnancy, with or without adjustment for the contribution of the gravid uterus.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 18750 ppm, food consumption was lower than control during the first two days of dietary exposure (Days 3-5) probably reflecting an initial reluctance to eat the treated diet due to its palatability. Thereafter, food intake during pregnancy was considered to be similar to control. There was no obvious effect on food intake during pregnancy at 3250 and 8750 ppm. At 8750 ppm, higher food intake attained statistical significance when compared with control but, in isolation, this finding was considered incidental and unrelated to treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was unaffected by dietary exposure at 3250, 8750 and 18750 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were apparent for females during the study.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.
Details on maternal toxic effects:
Pre-implantation loss, the number of implantations, subsequent embryofoetal survival and litter size and sex ratio on gestation day 20 was unaffected by maternal dietary exposure at 3250, 8750 or 18750 ppm.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
8 750 ppm
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 18750 ppm, mean foetal weights and litter weight were lower than control, with differences from control attaining statistical significance. Mean placental and total placenta weights were also slightly lower than control but only values for total placental weight attained statistical significance.

At 8750 ppm, mean litter and placental weights wereconsidered to be similar to control. Mean foetal weights (combined and female) were statistically lower than control.

At 3250 ppm, mean foetal, litter and placental weight were similar to control.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 18750 ppm, mean foetal weights and litter weight were lower than control, with differences from control attaining statistical significance. Mean placental and total placenta weights were also slightly lower than control but only values for total placental weight attained statistical significance.

At 8750 ppm, mean litter and placental weights wereconsidered to be similar to control. Mean foetal weights (combined and female) were statistically lower than control.

At 3250 ppm, mean foetal, litter and placental weight were similar to control.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during external examination of the foetuses at necropsy on gestation day 20 indicated any obvious effect of maternal treatment on foetal development at 3250, 8750 or 18750 ppm.

The incidence of foetuses with gastroschisis and encephalocoele at 18750 ppm, often also with spina bifida, was higher than control and was also above the historical control incidence. Although these findings were limited to one litter, an association with treatment cannot be discounted.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during skeletal examination of the foetuses indicated any obvious effect of maternal treatment on foetal development.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral examination of foetuses at 18750 ppm, revealed an apparent disturbance of the development of the kidney and ureter. This was principally manifested as increased incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla compared with control, although a low incidence of misshapen kidneys and absent renal medulla was also observed at this dietary level. The incidences of these findings were higher than observed for the historical control range.

At 8750 ppm, the incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla was also higher than control and the historical control range.

At 3250 ppm, only the incidence of foetuses/litters showing increased renal pelvic cavitation was higher than both concurrent control and the historical control range. The incidences of foetuses/litters showing kinked ureters or dilated ureters were similar to control and were within the historical control range.

The type, incidence and distribution of other visceral findings did not indicate any other effect of maternal treatment on foetal development at 3250, 8750 or 18750 ppm.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
3 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: urinary
Description (incidence and severity):
Cluster of visceral findings (kinked ureters, dilated ureters, increased renal pelvic cavitation, absent renal papilla misshapen kidneys and absent renal medulla) observed at the 8750 and 18750 ppm concentration levels.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
8 750 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 5 of Gestation

4

5

8

11

14

17

20

 

0 (Control)

Mean

5.2

9.4

19.3

40.6

54.3

86.5

137.6

SD

7.5

7.6

8.8

10.3

10.8

13.7

20.4

n

23

23

23

23

23

23

23

 

 

3250

Mean

4.6

8.4

18.4

41.1

55.0

85.7

134.3

SD

7.4

6.3

8.3

9.6

10.5

12.8

17.3

n

23

23

23

23

23

23

23

 

 

8750

Mean

2.5

8.8

20.8

43.0

57.8

87.2

135.5

SD

6.9

7.5

9.9

10.1

11.2

14.7

21.3

n

24

24

24

24

24

24

24

 

 

18750

Mean

-3.9***

1.6**

12.0*

35.7

52.3

78.1

122.9

SD

6.9

8.0

10.0

8.8

11.8

13.9

18.6

n

23

23

23

23

23

23

23

* 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

21.4

20.8

23.1

23.9

26.0

26.1

SD

4.9

2.6

2.2

3.3

2.7

3.4

n

23

23

23

23

23

23

 

 

3250

Mean

20.9

21.2

24.0

25.0

25.3

26.1

SD

3.1

3.0

2.7

2.1

2.0

2.3

n

23

23

23

23

23

23

 

 

8750

Mean

20.6

22.1

23.8

26.3*

26.3

26.2

SD

4.8

2.5

4.2

2.8

2.2

3.4

n

24

24

24

24

24

24

 

 

18750

Mean

17.5*

19.0

22.9

25.6

25.4

25.2

SD

2.6

2.9

2.8

3.1

2.8

2.3

n

23

23

23

23

23

23

* 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)

Total Placental Weight

(g)

 

0 (Control)

Mean

4.091

3.906

3.994

7.576

SD

0.218

0.199

0.200

1.317

n

23

23

23

23

 

 

3250

Mean

3.949

3.766

3.861

8.133

SD

0.299

0.304

0.314

1.493

n

23

23

23

23

 

 

8750

Mean

3.960

3.781*

3.865*

7.418

SD

0.275

0.260

0.263

1.409

n

24

24

24

24

 

 

18750

Mean

3.765**

(3.849**)

3.596**

(3.683*)

3.671**

(3.755**)

6.894*

(7.135*)

SD

0.461

(0.233)

0.495

(0.268)

0.472

(0.251)

1.715

(1.298)

n

23

(22)

23

(22)

23

(22)

23

(22)

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

( ) = Excludes one litter with atypically low foetal weights and significant foetal abnormalities.

Table 5. Summary Incidence of Foetal Visceral Findings

 

 

Visceral Findings

Dietary Concentration (ppm)

0 (Control)

3250

8750

18750

Number of Foetuses (litters) Examined

157 (23)

165 (23)

169 (24)

151 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Abdomen

Ureter – kinked

24

14

15.2

27

10

17.4

50

19

32.0*

60

19

38.6**

Ureter - dilated - Slight/Severe

18

10

11.3

16

9

10.8

36

16

24.1

48

16

31.1*

Renal pelvic cavitation - increased -

Slight/Severe

10

8

6.3

24

13

14.1

26

11

18.4

47

18

30.0***

Renal papilla – absent

1

1

0.9

0

0

0.0

6

4

5.9

13

6

9.0*

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, the No Observed Adverse Effect Level (NOAEL) for the pregnant rat was considered to be 8750 ppm (equivalent to 715.0 mg/kg bw/day) and the NOAEL for the developing conceptus was considered to be 3250 ppm (equivalent to 266.7 mg/kg bw/day).
Executive summary:

In a key pre-natal developmental toxicity study, the test material (Rosin, esters with ethylene glycol; CAS# 68512-65-2), 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 3250, 8750, and 18750 ppm (equivalent to a mean achieved dosage of 266.7, 715.0 and 1487.0 mg/kg bw/day respectively). 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 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.

 

No mortality or signs of clinical toxicity were observed through the study period. Initial dietary exposure at 18750 ppm was associated with a mean body weight loss, thereafter body weight gain was similar to control to gestation day 14. Lower body weight gain was observed from gestation days 14 to 20. During the later stage of gestation, body weight gain in the high exposure group may have been influenced by lower foetal/litter weight, however overall body weight remained lower than control, when adjusted for the contribution of the gravid uterus. Food consumption at this dietary level was lower than control during the first two days of dietary exposure, which probably reflected an initial reluctance to eat the treated diet (due to its palatability), but subsequent food intake was similar to control.

 

For litters at 18750 ppm, there was no effect of maternal exposure on pre-implantation loss, implantation count, embryofoetal survival, litter size or sex ratio but mean foetal, litter and placental weight were all lower than control. External examination of the foetuses at necropsy and subsequent detailed skeletal evaluation did not indicate any effect of maternal exposure on foetal development. However, there was a cluster of visceral findings (kinked ureters, dilated ureters, increased renal pelvic cavitation, absent renal papilla misshapen kidneys and absent renal medulla) that indicated a treatment-related disturbance of the normal development of the kidneys and ureters. One litter at 18750 ppm showed five foetuses with gastroschisis and encephalocoele. Despite the limitation of this finding to one litter, the group mean incidence of affected foetuses was higher than both concurrent and historical control values. As this finding was observed in the highest treated group an association with treatment cannot be discounted resulting in confirmation that dietary exposure at 18750 ppm is regarded as an adverse effect level for offspring development.

 

At 3250 and 8750 ppm, there was no effect of dietary exposure on maternal body weight gain or food consumption and pre-implantation loss, implantation count, embryofoetal survival, litter size, sex ratio and mean foetal, litter and placental weight were also considered to be unaffected by maternal exposure at these dietary levels. However, at 8750 ppm, the incidence of foetuses/litters showing kinked ureters, dilated ureters, increased renal pelvic cavitation and absent renal papilla was higher than control and the historical control range, indicating a treatment-related effect on the normal development of the kidneys and ureters. At 3250 ppm, the incidences of foetuses/litters showing kinked ureters or dilated ureters were similar to control and were within the historical control range. However, the incidence of foetuses/litters showing increased renal pelvic cavitation was higher than both concurrent control and the historical control range. While this higher incidence of increased renal pelvic cavitation was only slightly higher than the historical control range, in view of the findings apparent at higher dietary levels, an association with treatment cannot be discounted. However, increased renal pelvic cavitation is frequently observed amongst neonatal and adult animals within this laboratory without any obvious adverse impact of the viability of these animals and may be regarded, to a certain extent, as a variation of the normal. As this finding occurred in the absence of any increased incidence of more significant findings with the potential to effect offspring viability, such as absent renal papilla, and in the absence of any effect on skeletal development, this dietary level could be regarded as a No Observed Adverse Effect Level (NOAEL) for foetal development.

 

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for the pregnant rat was considered to be 8750 ppm (equivalent to 715 mg/kg bw/day) and the NOAEL for developmental toxicity was considered to be 3250 ppm (equivalent to 266.7 mg/kg bw/day).