Registration Dossier
Registration Dossier
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EC number: 294-402-5 | CAS number: 91722-01-9
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-08-26 to 2015-12-21
- 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 for cross-reference:
- read-across: supporting information
Reference
- Endpoint:
- developmental toxicity
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 2015-08-26 to 2015-12-21
- 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 for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- 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. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD®(SD) IGS BR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: Not specified
- Weight at study initiation: 186 to 277g
- 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 ± 3ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelvehours darkness
IN-LIFE DATES: From: 2015-08-28 To: 2016-09-16 - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures prepared in one batch and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous.This pre-mix was then added to a larger amount of basal laboratorydiet and mixed for a further sixty 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 determined as part of this study by the Test Facility. Representative samples were taken from the dietary admixtures and analyzed for concentration of Rosin, maleated CAS 8050-28-0 by the Test Facility. The results indicate that the prepared formulations were within 105 % to 107 % 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 Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited,Margate, Kent. Animals were delivered in two batches containing females at gestation Day 0 or 1 of treatment. 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 gestation day 19 inclusive.
- Frequency of treatment:
- continuously in the diet
- Duration of test:
- From gestation day 3 to gestation day 19 inclusive.
- Dose / conc.:
- 0 ppm
- Remarks:
- Control
- Dose / conc.:
- 500 ppm
- Remarks:
- Low Concentration
- Dose / conc.:
- 1 500 ppm
- Remarks:
- Intermediate Concentration
- Dose / conc.:
- 3 000 ppm
- Remarks:
- High Concentration
- No. of animals per sex per dose:
- 24 females/concentration
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Dose levels were selected in collaboration with the Sponsor based on available toxicity data including a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat (Harlan (Switzerland) Study Number D81040) for this route of administration. Dietary concentrations of 0 (Control), 500, 1500 and 3000 ppm were selected for use on this Pre-Natal Developmental Toxicity Study.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. - 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 (Day 20).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each individual animal on gestation days 3, 5, 8, 11, 14, 17 and 20.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on gestation day 20. All animals were subjected toa 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 uterus of the apparently non-pregnant female was 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 / No / No data
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 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 - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were apparent for adult females throughout the study at dietary concentrations of 500, 1500 or 3000 ppm of the test item.
- 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 exposed to a dietary concentration of 3000 ppm of the test item, a marginal mean body weight loss was apparent during the first day of dietary exposure, although the difference from controls failed to attain statistical significance. Subsequent body weight gains were frequently lower than control throughout gestation with differences attaining statistical significance during gestation days 8 to 11, 14 to 17 and 17 to 20. The inferior body weight gain, compared to control, was demonstrated more clearly by cumulative body weight gain from the start of treatment which was statistically significantly lower than control from gestation day 5. While body weight gain during late gestation was influenced by lower litter weight at this dietary level, final body weight and overall body weight gain when adjusted for the contribution of the gravid uteruswas still statistically significantly lower than control.
For females exposed to a dietary concentration of 1500 ppm of the test item, lower body weight gain during late gestation (gestation days 17 to 20) was statistically significantly lower than control. Prior to this, no statistically significant difference in body weight gain had been apparent and these differences in late gestation may reflect lower foetal weight growth at this dietary level. Supporting this, there were no statistically significant differences from control apparent for cumulative body weight gain, including overall body weight gain when adjusted for the contribution of the gravid uterus.
No statistically significant differences in body weight gain during gestation were apparent for females exposed to a dietary concentration of 500 ppm of the test item. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- For females exposed to a dietary concentration of 3000 ppm of the test item, food consumption was statistically significantly lower than control throughout gestation.
For females exposed to a dietary concentration of 500 or 1500 ppm of the test item, there was no obvious effect on food consumption during gestation. - Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Daily visual inspection of water bottles did not reveal any overt intergroup differences at 500, 1500 or 3000 ppm of the test item.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No macroscopic abnormalities were detected at necropsy on gestation day 20 in adult animals exposed to 500, 1500 or 3000 ppm of the test item.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Other effects:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect on live litter size or sex ratio.
- Details on maternal toxic effects:
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
Maternal dietary exposure to 3000 ppm of the test item resulted in lower numbers of early and total foetal deaths and lower post implantation loss attained statistical significance when compared with control. At 1500 ppm of the test item, lower numbers of total foetal deaths and lower post implantation loss also attained statistical significance when compared with control. These differences compared with control were considered to be incidental and unrelated to maternal dietary exposure.
The number of implantations and total number of live offspring was statistically significantly higher than control for dams exposed to 500 ppm of the test item. This was principally due to a higher corpora lutea count (an event established prior to treatment) and marginally superior foetal survival compared to control. This superior performance compared with control was considered to be incidental and unrelated to maternal dietary exposure to the test item. - Dose descriptor:
- NOEL
- Effect level:
- 500 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 500 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- 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): In offspring from dams exposed to 3000 ppm of the test item, mean foetal, litter and placental weights were statistically significantly lower than control.
In offspring from dams exposed to 1500 ppm of the test item, mean foetal and placental weights were statistically significantly lower thancontrol. Litter weight and total placental weight were also lower than control but differencesfailed to attain statistical significance.
At the 500 ppm maternal dietary exposure level there were no statistically significant differences in mean foetal and placental weights compared to control. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- In offspring from dams exposed to 3000 ppm of the test item, mean foetal, litter and placental weights were statistically significantly lower than control.
In offspring from dams exposed to 1500 ppm of the test item, mean foetal and placental weights were statistically significantly lower thancontrol. Litter weight and total placental weight were also lower than control but differencesfailed to attain statistical significance.
At the 500 ppm maternal dietary exposure level there were no statistically significant differences in mean foetal and placental weights compared to control. - External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At both the 1500 and 3000 ppm maternal dietary exposure levels, there was a higher incidence of visually small foetuses observed during the initial external fetal examination compared to control. Although the incidence of small foetuses failed to attain statistical significance, lower mean foetal weights were apparent for litters from the 1500 and 3000 ppm dietary levels. The incidence of other findings at either dietary exposure level did not indicate any obvious effect of maternal exposure to the test item on foetal growth or development.
At the 500 ppm maternal dietary exposure level, the incidence of external macroscopic findings did not indicate any obvious effect of maternal exposure to the test item on foetal growth or development. - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At the 3000 ppm maternal dietary exposure level there was a clear effect of maternal exposure to the test item on a large number ofossification parameters affecting most regions of the skeleton, with the number of foetuses/litters affected being increased compared with control and differences in foetal incidence frequently attaining statistical significance.
In the skull, these parameters included incomplete ossification of the nasal, frontal, parietal, interparietal, occipital squamosal, jugal, zygomatic process of the maxilla, zygomatic process of the squamosal, premaxilla, mandible, presphenoid and alisphenoid bones, unossified areas of the occipital bones and unossified hyoid bones. Additionally there was also a lower incidence of incomplete ossification of the basiophenoid bones.
In the vertebral column, these parameters included incomplete ossification of the cervical, lumbar and sacral (neural) arches and less than four ossified caudal vertebrae. Additionally there was a lower incidence of ossification present in the ventral arch of the first vertebra.
In the ribs and sternebrae, these parameters includes incomplete ossification of the ribs and sternebrae, wavy ribs, thickened ribs and misaligned costal cartilage. Additionally there was also a lower incidence of costal cartilage not fused to the sternebrae.
For the remaining skeleton, these parameters included incomplete ossification of the ischium, pubis and metacarpels and unossified metacarpels. Additionally there was also a lower incidence of ossified forepaw phalanges.
At the 1500 ppm maternal dietary exposure level there was a similar effect of maternal exposure to the test item on ossification parameters, although at a lower incidence than observed in the 3000 ppm dietary exposure group. The number of foetuses/litters affected were generally increased compared with control and differences in foetal incidence frequently attained statistical significance.
In the skull, these parameters included incomplete ossification of the nasal, frontal, parietal, interparietal, occipital squamosal, jugal, zygomatic process of the maxilla, zygomatic process of the squamosal, premaxilla and presphenoid bones and unossified areas of the occipital bones. Additionally there was also a lower incidence of incomplete ossification of the basiophencid bones.
In the vertebral column, these parameters included incomplete ossification of the cervical and sacral (neural) arches.
In the ribs and sternebrae, these parameters include incomplete ossification of the ribs, wavy ribs, thickened ribs and misaligned costal cartilage.
In the remaining skeleton, these parameters included incomplete ossification of the ischium; additionally there was also a lower incidence of ossified forepaw phalanges.
Neither the type, incidence nor distribution of skeletal findings observed in foetuses from dams exposed via the diet to 500 ppm of the test item indicated any effect of maternal exposure at that level. - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- Neither the type, incidence or distribution of visceral findings observed for foetuses indicate any effect of maternal exposure to the test item at 500, 1500 or 3000 ppm.
At the 1500 and 3000 ppm dietary exposure levels, the incidence of foetuses showing nonuniform patterning of the rugae was statistically significantly lower than control. However neither the incidence of affected foetuses or litters showed a clear relationship to exposure and, in isolation, this finding was considered unlikely to indicate an adverse effect on foetal development. - Dose descriptor:
- NOAEL
- Remarks:
- and NOEL
- Effect level:
- 500 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Developmental Toxicity
- Abnormalities:
- effects observed, treatment-related
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 500 ppm
- Treatment related:
- yes
- Relation to maternal toxicity:
- not specified
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Based on the results of this study, dietary exposure to 500 ppm of the test item (equivalent to an achieved dosage of approximately 41.2 mg/kg bw/day of the test material) is considered to be a clear No Observed Effect Level (NOEL) for the pregnant dam. The No Observed Adverse Effect Level (NOAEL) for the pregnant dam was considered to be a dietary exposure to 1500 ppm of the test material (equivalent to an achieved dosage of approximately 122.9 mg/kg bw/day of the test item) due to effects on body weight gain and food consumption apparent at a dietary level of 3000 ppm.
The NOEL for the developing conceptus was considered to be a maternal dietary exposure to 500 ppm of the test material. The NOAEL for the developing conceptus was also considered to be at least 500 ppm of the test material, due to effects on foetal and placental weights and numerous skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton at the 1500 ppm level. - Executive summary:
In a key pre-natal developmental toxicity study, the test material (Rosin, maleated; CAS# 8050-28-0) was administered by continuous dietary admixture to three groups each composed of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between gestation days 3 and 19 (inclusive) at dietary concentrations of 500, 1500, and 3000 ppm equivalent to mean achieved dosages of approximately 41.2, 122.9 and 217.4 mg/kg bw/day. 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 weights, sex and external and internal macroscopic appearances were recorded. Half of the pups of each litter were examined for detailed
skeletal development and the remainder were subjected to detailed visceral examination.
No mortality was and no clinical signs were apparent for adult females throughout the study at dietary concentrations of 500, 1500 or 3000 ppm of the test material.
At a dietary concentration of 3000 ppm of the test material, marginal mean body weight loss was apparent during the first day of dietary exposure and subsequent bodyweight gains were often statistically significantly lower than control animals throughout gestation. Cumulative body weight gain from the start of treatment was statistically significantly lower than control animals from gestation day 5 and overall body weight gain, when adjusted for the contribution of the gravid uterus, remained statistically significantly lower than control. In rats exposed to 1500 ppm of the test material, statistically significantly lower body weight gain was apparent during gestation days 17 to 20. Prior to this, no statistically significant difference in body weight gain had been apparent and there were no statistically significant differences from control animals apparent for cumulative body weight gain, including overall body weight gain when adjusted for the contribution of the gravid uterus. No statistically significant differences in body weight gain during gestation were apparent for females that received 500 ppm of the test material. Food consumption throughout gestation was statistically significantly lower in rats exposed to the test material at 3000 ppm when compared with control animals. No obvious effects on food consumption during gestation were observed in animals exposed to the test material at 500 or 1500 ppm.
Gross necropsy on Gestation Day 20, did not reveal any remarkable findings (macroscopic abnormalities) in adult animals in any of the treatment groups.
There was no effect of maternal dietary exposure 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 at dietary exposures of 500, 1500 or 3000 ppm of the test material.
Maternal dietary exposure to 3000 ppm of the test material resulted in statistically significantly lower mean foetal, litter and placental weights when compared to the control animals. At the 1500 ppm maternal dietary exposure level, mean foetal and placental weights were statistically significantly lower than the control group. Litter weight and total placental weight were also lower when compared to the control group, but differences failed to attain statistical significance. No statistically significant differences in mean foetal and placental weights were observed in the 500 ppm treatment group when compared to the control animals.
External examination at necropsy revealed a higher incidence of visually small foetuses in the 3000 ppm and, to a lesser extent, the 1500 ppm maternal dietary exposure groups, when compared to the control. No such effects were apparent in the 500 ppm treatment group. Neither the type, incidence nor the distribution of visceral findings observed in the foetuses indicated any effect of maternal dietary exposure to 500, 1500 or 3000 ppm of the test material.
Maternal dietary exposure to 3000 ppm of the test material, resulted in a clear effect on a large number of ossification parameters affecting most regions of the skeleton. There was (generally) an increase in both the number of foetuses and litters affected compared to the control group and statistically significant differences from control in foetal incidence for these parameters. At the 1500 ppm maternal dietary exposure level, there was a similar effect on ossification parameters, although changes were not quite as extensive and generally occurred at a lower incidence than that observed at the 3000 ppm dietary exposure level. Again, there was generally an increase in both the number of foetuses and litters affected compared to the control group and statistically significant differences from control in foetal incidence for these parameters. Neither the type, incidence nor the distribution of skeletal findings observed for foetuses indicated any effect of maternal dietary exposure to 500 ppm of the test item.
Based on the results of this study, dietary exposure to 500 ppm of the test material (equivalent to an achieved dosage of approximately 41.2 mg/kg bw/day of the test material) was considered to be a clear No Observed Effect Level (NOEL) for the pregnant dam. The No Observed Adverse Effect Level (NOAEL) for the pregnant dam was considered to be a dietary exposure to 1500 ppm of the test item (equivalent to an achieved dosage of approximately 122.9 mg/kg bw/day of the test material), due to effects on body weight gain and food consumption apparent at a dietary level of 3000 ppm.
The NOEL for the developing conceptus was considered to be a maternal dietary exposure to 500 ppm of the test item. The NOAEL for developmental toxicity was also considered to be at least 500 ppm of the test material, due to effects on foetal and placental weights and numerous skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton at the 1500 ppm level.
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 |
4.3 |
8.1 |
24.0 |
43.8 |
60.4 |
91.2 |
137.3 |
SD |
9.2 |
7.2 |
9.8 |
9.8 |
11.6 |
12.4 |
16.8 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
||||||||
500 |
Mean |
1.6 |
8.3 |
23.8 |
43.7 |
60.8 |
91.2 |
138.7 |
SD |
7.0 |
7.4 |
9.0 |
8.5 |
11.5 |
13.3 |
18.9 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
||||||||
1500 |
Mean |
1.5 |
10.0 |
25.5 |
42.5 |
59.0 |
87.6 |
127.3 |
SD |
7.9 |
5.4 |
7.7 |
8.0 |
13.5 |
14.0 |
16.6 |
|
n |
23 |
23 |
23 |
23 |
23 |
23 |
23 |
|
|
||||||||
3000 |
Mean |
-1.1 |
2.5* |
14.1** |
27.5*** |
43.7*** |
66.8*** |
103.7*** |
SD |
6.6 |
5.7 |
10.7 |
11.9 |
14.5 |
15.3 |
17.9 |
|
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 |
17.6 |
20.5 |
22.8 |
24.1 |
26.6 |
27.0 |
SD |
3.8 |
3.0 |
2.5 |
2.2 |
2.5 |
2.7 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
500 |
Mean |
17.7 |
20.4 |
24.1 |
24.6 |
26.4 |
25.7 |
SD |
2.8 |
2.9 |
2.3 |
2.2 |
2.1 |
2.4 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
1500 |
Mean |
17.0 |
20.5 |
22.8 |
24.0 |
26.0 |
25.4 |
SD |
2.9 |
2.3 |
2.0 |
2.5 |
2.4 |
2.6 |
|
n |
23 |
23 |
23 |
23 |
23 |
23 |
|
|
|||||||
3000 |
Mean |
13.1*** |
17.3*** |
19.0*** |
20.5*** |
22.4*** |
22.4*** |
SD |
2.5 |
2.5 |
2.5 |
2.5 |
2.3 |
2.3 |
|
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) |
Mean Placental Weight (g) |
Litter Weight (g) |
Total Placental Weight (g) |
0 (Control) |
Mean |
4.159 |
3.987 |
4.064 |
0.611 |
54.159 |
8.095 |
SD |
0.211 |
0.229 |
0.210 |
0.097 |
8.229 |
1.508 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
500 |
Mean |
4.001 |
3.868 |
3.932 |
0.572 |
58.418 |
8.488 |
SD |
0.175 |
0.203 |
0.184 |
0.073 |
7.654 |
1.422 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
1500 |
Mean |
3.744*** |
3.552*** |
3.650*** |
0.534** |
49.406 |
7.238 |
SD |
0.234 |
0.254 |
0.235 |
0.055 |
7.808 |
1.353 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
3000 |
Mean |
3.512*** |
3.311*** |
3.413*** |
0.516** |
47.158** |
7.143* |
SD |
0.299 |
0.304 |
0.298 |
0.050 |
4.687 |
0.864 |
|
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 5. Summary Incidence of Foetal Skeletal Findings |
||||||||||||||
Skeletal Findings |
Dietary Concentration (ppm) |
|||||||||||||
0 (Control) |
500 |
1500 |
3000 |
|||||||||||
Number of Foetuses (litters) Examined |
||||||||||||||
165 (24) |
184 (24) |
168 (24) |
166 (23) |
|||||||||||
NF |
NL |
%† |
NF |
NL |
%† |
NF |
NL |
%† |
NF |
NL |
%† |
|||
Head |
||||||||||||||
Rugae - non-uniform patterning |
24 |
15 |
14.1 |
17 |
13 |
9.4 |
9 |
7 |
5.1* |
10 |
8 |
6.1* |
||
Skull |
||||||||||||||
Nasal - incomplete ossification |
6 |
5 |
4.0 |
14 |
9 |
7.5 |
21 |
11 |
13.1* |
58 |
17 |
37.6*** |
||
Frontal - incomplete ossification |
2 |
2 |
1.4 |
5 |
5 |
2.8 |
15 |
10 |
8.9** |
58 |
19 |
37.6*** |
||
Parietal - incomplete ossification |
7 |
6 |
4.5 |
22 |
13 |
12.3 |
30 |
16 |
18.7** |
90 |
21 |
59.2*** |
||
Interparietal - incomplete ossification |
21 |
10 |
13.0 |
38 |
16 |
21.6 |
64 |
19 |
39.3** |
121 |
23 |
79.1*** |
||
Occipital (Supra-occipital) - incomplete ossification |
19 |
9 |
11.9 |
23 |
10 |
13.1 |
59 |
18 |
37.3** |
122 |
23 |
79.5*** |
||
Squamosal - incomplete ossification |
17 |
10 |
10.3 |
35 |
16 |
19.7 |
53 |
17 |
32.7** |
109 |
22 |
71.4*** |
||
Jugal - incomplete ossification |
11 |
9 |
7.5 |
17 |
9 |
9.8 |
31 |
16 |
19.6* |
66 |
19 |
43.5*** |
||
Zygomatic process of maxilla - incomplete ossification |
7 |
5 |
5.3 |
22 |
12 |
11.9 |
26 |
15 |
16.6** |
65 |
21 |
41.6*** |
||
Zygomatic process of squamosal - incomplete ossification |
2 |
2 |
1.2 |
6 |
3 |
3.2 |
11 |
6 |
7.1 |
45 |
15 |
29.8*** |
||
Premaxilla - incomplete ossification |
0 |
0 |
0.0 |
2 |
2 |
1.2 |
8 |
7 |
5.5** |
29 |
12 |
18.6*** |
||
Hyoid - not ossified |
23 |
14 |
14.8 |
14 |
11 |
7.8 |
27 |
15 |
16.8 |
50 |
21 |
32.3** |
||
Mandible - incomplete ossification |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
23 |
7 |
14.5** |
||
Presphenoid - incomplete ossification |
2 |
2 |
1.4 |
3 |
3 |
1.6 |
11 |
6 |
6.8 |
42 |
14 |
27.6*** |
||
Alisphenoid - incomplete ossification |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
12 |
8 |
7.9** |
||
Basisphenoid - incomplete ossification |
4 |
4 |
2.9 |
1 |
1 |
0.7 |
0 |
0 |
0.0* |
0 |
0 |
0.0* |
||
Vertebral Column |
||||||||||||||
Ventral arch of vertebra 1 - ossification present |
42 |
18 |
27.2 |
45 |
18 |
25.4 |
36 |
16 |
22.4 |
13 |
7 |
8.8** |
||
Cervical (neural) arch - incomplete ossification |
7 |
3 |
4.4 |
9 |
7 |
4.8 |
17 |
13 |
10.7** |
60 |
20 |
38.6*** |
||
Lumbar (neural) arch - incomplete ossification |
1 |
1 |
0.8 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
35 |
15 |
23.3*** |
||
Sacral (neural) arch - incomplete ossification |
14 |
9 |
9.0 |
36 |
12 |
21.5 |
46 |
17 |
29.4** |
71 |
21 |
45.6*** |
||
Caudal vertebrae - less than 4 ossified |
28 |
11 |
17.9 |
31 |
12 |
18.0 |
45 |
17 |
28.8 |
77 |
19 |
50.1** |
||
Ribs |
||||||||||||||
One or more ribs – wavy |
0 |
0 |
0.0 |
3 |
1 |
1.6 |
7 |
6 |
4.5* |
52 |
19 |
34.2*** |
||
One or more ribs – thickened |
1 |
1 |
0.8 |
2 |
1 |
1.0 |
14 |
11 |
9.0** |
44 |
19 |
29.1*** |
||
Rib - incomplete ossification |
0 |
0 |
0.0 |
2 |
1 |
1.0 |
4 |
4 |
2.3* |
37 |
15 |
24.8*** |
||
Costal cartilage – misaligned |
2 |
2 |
1.9 |
4 |
4 |
2.2 |
4 |
3 |
2.5 |
10 |
10 |
6.7* |
||
Costal cartilage - not fused to sternebra |
31 |
17 |
21.3 |
8 |
6 |
4.8 |
15 |
7 |
10.1 |
11 |
8 |
7.0* |
||
Sternebrae |
||||||||||||||
Sternebra - incomplete ossification |
3 |
1 |
1.6 |
2 |
1 |
1.0 |
8 |
4 |
4.3 |
10 |
7 |
6.3* |
||
Pelvic Girdle |
||||||||||||||
Femur - incomplete ossification |
2 |
2 |
1.4 |
7 |
4 |
3.7 |
12 |
8 |
7.4* |
35 |
15 |
22.8*** |
||
Pubis - incomplete ossification |
7 |
5 |
4.4 |
11 |
8 |
6.2 |
21 |
11 |
13.4 |
41 |
17 |
26.7*** |
||
Forelimbs |
||||||||||||||
Metacarpal - not ossified |
44 |
18 |
28.0 |
38 |
15 |
22.1 |
61 |
19 |
38.7 |
106 |
22 |
68.7*** |
||
Metacarpal - incomplete ossification |
2 |
2 |
1.4 |
4 |
4 |
2.2 |
6 |
5 |
4.1 |
25 |
15 |
16.4*** |
||
Forepaw phalanges - 1 or more – ossified |
28 |
14 |
17.9 |
33 |
13 |
20.2 |
10 |
6 |
6.7* |
8 |
3 |
5.3** |
||
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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- 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. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Rosin maleated
- IUPAC Name:
- Rosin maleated
- Test material form:
- solid
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD®(SD) IGS BR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: Not specified
- Weight at study initiation: 186 to 277g
- 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 ± 3ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelvehours darkness
IN-LIFE DATES: From: 2015-08-28 To: 2016-09-16
Administration / exposure
- Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures prepared in one batch and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous.This pre-mix was then added to a larger amount of basal laboratorydiet and mixed for a further sixty 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 determined as part of this study by the Test Facility. Representative samples were taken from the dietary admixtures and analyzed for concentration of Rosin, maleated CAS 8050-28-0 by the Test Facility. The results indicate that the prepared formulations were within 105 % to 107 % 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 Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited,Margate, Kent. Animals were delivered in two batches containing females at gestation Day 0 or 1 of treatment. 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 gestation day 19 inclusive.
- Frequency of treatment:
- continuously in the diet
- Duration of test:
- From gestation day 3 to gestation day 19 inclusive.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 ppm
- Remarks:
- Control
- Dose / conc.:
- 500 ppm
- Remarks:
- Low Concentration
- Dose / conc.:
- 1 500 ppm
- Remarks:
- Intermediate Concentration
- Dose / conc.:
- 3 000 ppm
- Remarks:
- High Concentration
- No. of animals per sex per dose:
- 24 females/concentration
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: Dose levels were selected in collaboration with the Sponsor based on available toxicity data including a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat (Harlan (Switzerland) Study Number D81040) for this route of administration. Dietary concentrations of 0 (Control), 500, 1500 and 3000 ppm were selected for use on this Pre-Natal Developmental Toxicity Study.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: 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 (Day 20).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each individual animal on gestation days 3, 5, 8, 11, 14, 17 and 20.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on gestation day 20. All animals were subjected toa 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 uterus of the apparently non-pregnant female was 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 / No / No data
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 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
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs were apparent for adult females throughout the study at dietary concentrations of 500, 1500 or 3000 ppm of the test item.
- 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 exposed to a dietary concentration of 3000 ppm of the test item, a marginal mean body weight loss was apparent during the first day of dietary exposure, although the difference from controls failed to attain statistical significance. Subsequent body weight gains were frequently lower than control throughout gestation with differences attaining statistical significance during gestation days 8 to 11, 14 to 17 and 17 to 20. The inferior body weight gain, compared to control, was demonstrated more clearly by cumulative body weight gain from the start of treatment which was statistically significantly lower than control from gestation day 5. While body weight gain during late gestation was influenced by lower litter weight at this dietary level, final body weight and overall body weight gain when adjusted for the contribution of the gravid uteruswas still statistically significantly lower than control.
For females exposed to a dietary concentration of 1500 ppm of the test item, lower body weight gain during late gestation (gestation days 17 to 20) was statistically significantly lower than control. Prior to this, no statistically significant difference in body weight gain had been apparent and these differences in late gestation may reflect lower foetal weight growth at this dietary level. Supporting this, there were no statistically significant differences from control apparent for cumulative body weight gain, including overall body weight gain when adjusted for the contribution of the gravid uterus.
No statistically significant differences in body weight gain during gestation were apparent for females exposed to a dietary concentration of 500 ppm of the test item. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- For females exposed to a dietary concentration of 3000 ppm of the test item, food consumption was statistically significantly lower than control throughout gestation.
For females exposed to a dietary concentration of 500 or 1500 ppm of the test item, there was no obvious effect on food consumption during gestation. - Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Daily visual inspection of water bottles did not reveal any overt intergroup differences at 500, 1500 or 3000 ppm of the test item.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No macroscopic abnormalities were detected at necropsy on gestation day 20 in adult animals exposed to 500, 1500 or 3000 ppm of the test item.
Maternal developmental toxicity
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
- Other effects:
- no effects observed
- Description (incidence and severity):
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect on live litter size or sex ratio.
- Details on maternal toxic effects:
- Maternal dietary exposure to 500, 15000 or 3000 ppm of the test item produced no effect 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 or pre and postimplantation losses.
Maternal dietary exposure to 3000 ppm of the test item resulted in lower numbers of early and total foetal deaths and lower post implantation loss attained statistical significance when compared with control. At 1500 ppm of the test item, lower numbers of total foetal deaths and lower post implantation loss also attained statistical significance when compared with control. These differences compared with control were considered to be incidental and unrelated to maternal dietary exposure.
The number of implantations and total number of live offspring was statistically significantly higher than control for dams exposed to 500 ppm of the test item. This was principally due to a higher corpora lutea count (an event established prior to treatment) and marginally superior foetal survival compared to control. This superior performance compared with control was considered to be incidental and unrelated to maternal dietary exposure to the test item.
Effect levels (maternal animals)
open allclose all
- Dose descriptor:
- NOEL
- Effect level:
- 500 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 1 500 ppm
- Based on:
- test mat.
- Basis for effect level:
- other: Systemic Toxicity
Maternal abnormalities
- 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): In offspring from dams exposed to 3000 ppm of the test item, mean foetal, litter and placental weights were statistically significantly lower than control.
In offspring from dams exposed to 1500 ppm of the test item, mean foetal and placental weights were statistically significantly lower thancontrol. Litter weight and total placental weight were also lower than control but differencesfailed to attain statistical significance.
At the 500 ppm maternal dietary exposure level there were no statistically significant differences in mean foetal and placental weights compared to control. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- In offspring from dams exposed to 3000 ppm of the test item, mean foetal, litter and placental weights were statistically significantly lower than control.
In offspring from dams exposed to 1500 ppm of the test item, mean foetal and placental weights were statistically significantly lower thancontrol. Litter weight and total placental weight were also lower than control but differencesfailed to attain statistical significance.
At the 500 ppm maternal dietary exposure level there were no statistically significant differences in mean foetal and placental weights compared to control. - External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At both the 1500 and 3000 ppm maternal dietary exposure levels, there was a higher incidence of visually small foetuses observed during the initial external fetal examination compared to control. Although the incidence of small foetuses failed to attain statistical significance, lower mean foetal weights were apparent for litters from the 1500 and 3000 ppm dietary levels. The incidence of other findings at either dietary exposure level did not indicate any obvious effect of maternal exposure to the test item on foetal growth or development.
At the 500 ppm maternal dietary exposure level, the incidence of external macroscopic findings did not indicate any obvious effect of maternal exposure to the test item on foetal growth or development. - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- At the 3000 ppm maternal dietary exposure level there was a clear effect of maternal exposure to the test item on a large number ofossification parameters affecting most regions of the skeleton, with the number of foetuses/litters affected being increased compared with control and differences in foetal incidence frequently attaining statistical significance.
In the skull, these parameters included incomplete ossification of the nasal, frontal, parietal, interparietal, occipital squamosal, jugal, zygomatic process of the maxilla, zygomatic process of the squamosal, premaxilla, mandible, presphenoid and alisphenoid bones, unossified areas of the occipital bones and unossified hyoid bones. Additionally there was also a lower incidence of incomplete ossification of the basiophenoid bones.
In the vertebral column, these parameters included incomplete ossification of the cervical, lumbar and sacral (neural) arches and less than four ossified caudal vertebrae. Additionally there was a lower incidence of ossification present in the ventral arch of the first vertebra.
In the ribs and sternebrae, these parameters includes incomplete ossification of the ribs and sternebrae, wavy ribs, thickened ribs and misaligned costal cartilage. Additionally there was also a lower incidence of costal cartilage not fused to the sternebrae.
For the remaining skeleton, these parameters included incomplete ossification of the ischium, pubis and metacarpels and unossified metacarpels. Additionally there was also a lower incidence of ossified forepaw phalanges.
At the 1500 ppm maternal dietary exposure level there was a similar effect of maternal exposure to the test item on ossification parameters, although at a lower incidence than observed in the 3000 ppm dietary exposure group. The number of foetuses/litters affected were generally increased compared with control and differences in foetal incidence frequently attained statistical significance.
In the skull, these parameters included incomplete ossification of the nasal, frontal, parietal, interparietal, occipital squamosal, jugal, zygomatic process of the maxilla, zygomatic process of the squamosal, premaxilla and presphenoid bones and unossified areas of the occipital bones. Additionally there was also a lower incidence of incomplete ossification of the basiophencid bones.
In the vertebral column, these parameters included incomplete ossification of the cervical and sacral (neural) arches.
In the ribs and sternebrae, these parameters include incomplete ossification of the ribs, wavy ribs, thickened ribs and misaligned costal cartilage.
In the remaining skeleton, these parameters included incomplete ossification of the ischium; additionally there was also a lower incidence of ossified forepaw phalanges.
Neither the type, incidence nor distribution of skeletal findings observed in foetuses from dams exposed via the diet to 500 ppm of the test item indicated any effect of maternal exposure at that level. - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- Neither the type, incidence or distribution of visceral findings observed for foetuses indicate any effect of maternal exposure to the test item at 500, 1500 or 3000 ppm.
At the 1500 and 3000 ppm dietary exposure levels, the incidence of foetuses showing nonuniform patterning of the rugae was statistically significantly lower than control. However neither the incidence of affected foetuses or litters showed a clear relationship to exposure and, in isolation, this finding was considered unlikely to indicate an adverse effect on foetal development.
Effect levels (fetuses)
- Dose descriptor:
- NOAEL
- Remarks:
- and NOEL
- Effect level:
- 500 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Developmental Toxicity
Fetal abnormalities
- Abnormalities:
- effects observed, treatment-related
Overall developmental toxicity
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 500 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 |
4.3 |
8.1 |
24.0 |
43.8 |
60.4 |
91.2 |
137.3 |
SD |
9.2 |
7.2 |
9.8 |
9.8 |
11.6 |
12.4 |
16.8 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
||||||||
500 |
Mean |
1.6 |
8.3 |
23.8 |
43.7 |
60.8 |
91.2 |
138.7 |
SD |
7.0 |
7.4 |
9.0 |
8.5 |
11.5 |
13.3 |
18.9 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
||||||||
1500 |
Mean |
1.5 |
10.0 |
25.5 |
42.5 |
59.0 |
87.6 |
127.3 |
SD |
7.9 |
5.4 |
7.7 |
8.0 |
13.5 |
14.0 |
16.6 |
|
n |
23 |
23 |
23 |
23 |
23 |
23 |
23 |
|
|
||||||||
3000 |
Mean |
-1.1 |
2.5* |
14.1** |
27.5*** |
43.7*** |
66.8*** |
103.7*** |
SD |
6.6 |
5.7 |
10.7 |
11.9 |
14.5 |
15.3 |
17.9 |
|
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 |
17.6 |
20.5 |
22.8 |
24.1 |
26.6 |
27.0 |
SD |
3.8 |
3.0 |
2.5 |
2.2 |
2.5 |
2.7 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
500 |
Mean |
17.7 |
20.4 |
24.1 |
24.6 |
26.4 |
25.7 |
SD |
2.8 |
2.9 |
2.3 |
2.2 |
2.1 |
2.4 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
1500 |
Mean |
17.0 |
20.5 |
22.8 |
24.0 |
26.0 |
25.4 |
SD |
2.9 |
2.3 |
2.0 |
2.5 |
2.4 |
2.6 |
|
n |
23 |
23 |
23 |
23 |
23 |
23 |
|
|
|||||||
3000 |
Mean |
13.1*** |
17.3*** |
19.0*** |
20.5*** |
22.4*** |
22.4*** |
SD |
2.5 |
2.5 |
2.5 |
2.5 |
2.3 |
2.3 |
|
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) |
Mean Placental Weight (g) |
Litter Weight (g) |
Total Placental Weight (g) |
0 (Control) |
Mean |
4.159 |
3.987 |
4.064 |
0.611 |
54.159 |
8.095 |
SD |
0.211 |
0.229 |
0.210 |
0.097 |
8.229 |
1.508 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
500 |
Mean |
4.001 |
3.868 |
3.932 |
0.572 |
58.418 |
8.488 |
SD |
0.175 |
0.203 |
0.184 |
0.073 |
7.654 |
1.422 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
1500 |
Mean |
3.744*** |
3.552*** |
3.650*** |
0.534** |
49.406 |
7.238 |
SD |
0.234 |
0.254 |
0.235 |
0.055 |
7.808 |
1.353 |
|
n |
24 |
24 |
24 |
24 |
24 |
24 |
|
|
|||||||
3000 |
Mean |
3.512*** |
3.311*** |
3.413*** |
0.516** |
47.158** |
7.143* |
SD |
0.299 |
0.304 |
0.298 |
0.050 |
4.687 |
0.864 |
|
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 5. Summary Incidence of Foetal Skeletal Findings |
||||||||||||||
Skeletal Findings |
Dietary Concentration (ppm) |
|||||||||||||
0 (Control) |
500 |
1500 |
3000 |
|||||||||||
Number of Foetuses (litters) Examined |
||||||||||||||
165 (24) |
184 (24) |
168 (24) |
166 (23) |
|||||||||||
NF |
NL |
%† |
NF |
NL |
%† |
NF |
NL |
%† |
NF |
NL |
%† |
|||
Head |
||||||||||||||
Rugae - non-uniform patterning |
24 |
15 |
14.1 |
17 |
13 |
9.4 |
9 |
7 |
5.1* |
10 |
8 |
6.1* |
||
Skull |
||||||||||||||
Nasal - incomplete ossification |
6 |
5 |
4.0 |
14 |
9 |
7.5 |
21 |
11 |
13.1* |
58 |
17 |
37.6*** |
||
Frontal - incomplete ossification |
2 |
2 |
1.4 |
5 |
5 |
2.8 |
15 |
10 |
8.9** |
58 |
19 |
37.6*** |
||
Parietal - incomplete ossification |
7 |
6 |
4.5 |
22 |
13 |
12.3 |
30 |
16 |
18.7** |
90 |
21 |
59.2*** |
||
Interparietal - incomplete ossification |
21 |
10 |
13.0 |
38 |
16 |
21.6 |
64 |
19 |
39.3** |
121 |
23 |
79.1*** |
||
Occipital (Supra-occipital) - incomplete ossification |
19 |
9 |
11.9 |
23 |
10 |
13.1 |
59 |
18 |
37.3** |
122 |
23 |
79.5*** |
||
Squamosal - incomplete ossification |
17 |
10 |
10.3 |
35 |
16 |
19.7 |
53 |
17 |
32.7** |
109 |
22 |
71.4*** |
||
Jugal - incomplete ossification |
11 |
9 |
7.5 |
17 |
9 |
9.8 |
31 |
16 |
19.6* |
66 |
19 |
43.5*** |
||
Zygomatic process of maxilla - incomplete ossification |
7 |
5 |
5.3 |
22 |
12 |
11.9 |
26 |
15 |
16.6** |
65 |
21 |
41.6*** |
||
Zygomatic process of squamosal - incomplete ossification |
2 |
2 |
1.2 |
6 |
3 |
3.2 |
11 |
6 |
7.1 |
45 |
15 |
29.8*** |
||
Premaxilla - incomplete ossification |
0 |
0 |
0.0 |
2 |
2 |
1.2 |
8 |
7 |
5.5** |
29 |
12 |
18.6*** |
||
Hyoid - not ossified |
23 |
14 |
14.8 |
14 |
11 |
7.8 |
27 |
15 |
16.8 |
50 |
21 |
32.3** |
||
Mandible - incomplete ossification |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
23 |
7 |
14.5** |
||
Presphenoid - incomplete ossification |
2 |
2 |
1.4 |
3 |
3 |
1.6 |
11 |
6 |
6.8 |
42 |
14 |
27.6*** |
||
Alisphenoid - incomplete ossification |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
0 |
0 |
0.0 |
12 |
8 |
7.9** |
||
Basisphenoid - incomplete ossification |
4 |
4 |
2.9 |
1 |
1 |
0.7 |
0 |
0 |
0.0* |
0 |
0 |
0.0* |
||
Vertebral Column |
||||||||||||||
Ventral arch of vertebra 1 - ossification present |
42 |
18 |
27.2 |
45 |
18 |
25.4 |
36 |
16 |
22.4 |
13 |
7 |
8.8** |
||
Cervical (neural) arch - incomplete ossification |
7 |
3 |
4.4 |
9 |
7 |
4.8 |
17 |
13 |
10.7** |
60 |
20 |
38.6*** |
||
Lumbar (neural) arch - incomplete ossification |
1 |
1 |
0.8 |
0 |
0 |
0.0 |
1 |
1 |
0.7 |
35 |
15 |
23.3*** |
||
Sacral (neural) arch - incomplete ossification |
14 |
9 |
9.0 |
36 |
12 |
21.5 |
46 |
17 |
29.4** |
71 |
21 |
45.6*** |
||
Caudal vertebrae - less than 4 ossified |
28 |
11 |
17.9 |
31 |
12 |
18.0 |
45 |
17 |
28.8 |
77 |
19 |
50.1** |
||
Ribs |
||||||||||||||
One or more ribs – wavy |
0 |
0 |
0.0 |
3 |
1 |
1.6 |
7 |
6 |
4.5* |
52 |
19 |
34.2*** |
||
One or more ribs – thickened |
1 |
1 |
0.8 |
2 |
1 |
1.0 |
14 |
11 |
9.0** |
44 |
19 |
29.1*** |
||
Rib - incomplete ossification |
0 |
0 |
0.0 |
2 |
1 |
1.0 |
4 |
4 |
2.3* |
37 |
15 |
24.8*** |
||
Costal cartilage – misaligned |
2 |
2 |
1.9 |
4 |
4 |
2.2 |
4 |
3 |
2.5 |
10 |
10 |
6.7* |
||
Costal cartilage - not fused to sternebra |
31 |
17 |
21.3 |
8 |
6 |
4.8 |
15 |
7 |
10.1 |
11 |
8 |
7.0* |
||
Sternebrae |
||||||||||||||
Sternebra - incomplete ossification |
3 |
1 |
1.6 |
2 |
1 |
1.0 |
8 |
4 |
4.3 |
10 |
7 |
6.3* |
||
Pelvic Girdle |
||||||||||||||
Femur - incomplete ossification |
2 |
2 |
1.4 |
7 |
4 |
3.7 |
12 |
8 |
7.4* |
35 |
15 |
22.8*** |
||
Pubis - incomplete ossification |
7 |
5 |
4.4 |
11 |
8 |
6.2 |
21 |
11 |
13.4 |
41 |
17 |
26.7*** |
||
Forelimbs |
||||||||||||||
Metacarpal - not ossified |
44 |
18 |
28.0 |
38 |
15 |
22.1 |
61 |
19 |
38.7 |
106 |
22 |
68.7*** |
||
Metacarpal - incomplete ossification |
2 |
2 |
1.4 |
4 |
4 |
2.2 |
6 |
5 |
4.1 |
25 |
15 |
16.4*** |
||
Forepaw phalanges - 1 or more – ossified |
28 |
14 |
17.9 |
33 |
13 |
20.2 |
10 |
6 |
6.7* |
8 |
3 |
5.3** |
||
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, dietary exposure to 500 ppm of the test item (equivalent to an achieved dosage of approximately 41.2 mg/kg bw/day of the test material) is considered to be a clear No Observed Effect Level (NOEL) for the pregnant dam. The No Observed Adverse Effect Level (NOAEL) for the pregnant dam was considered to be a dietary exposure to 1500 ppm of the test material (equivalent to an achieved dosage of approximately 122.9 mg/kg bw/day of the test item) due to effects on body weight gain and food consumption apparent at a dietary level of 3000 ppm.
The NOEL for the developing conceptus was considered to be a maternal dietary exposure to 500 ppm of the test material. The NOAEL for the developing conceptus was also considered to be at least 500 ppm of the test material, due to effects on foetal and placental weights and numerous skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton at the 1500 ppm level. - Executive summary:
In a key pre-natal developmental toxicity study, the test material (Rosin, maleated; CAS# 8050-28-0) was administered by continuous dietary admixture to three groups each composed of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between gestation days 3 and 19 (inclusive) at dietary concentrations of 500, 1500, and 3000 ppm equivalent to mean achieved dosages of approximately 41.2, 122.9 and 217.4 mg/kg bw/day. 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 weights, sex and external and internal macroscopic appearances were recorded. Half of the pups of each litter were examined for detailed
skeletal development and the remainder were subjected to detailed visceral examination.
No mortality was and no clinical signs were apparent for adult females throughout the study at dietary concentrations of 500, 1500 or 3000 ppm of the test material.
At a dietary concentration of 3000 ppm of the test material, marginal mean body weight loss was apparent during the first day of dietary exposure and subsequent bodyweight gains were often statistically significantly lower than control animals throughout gestation. Cumulative body weight gain from the start of treatment was statistically significantly lower than control animals from gestation day 5 and overall body weight gain, when adjusted for the contribution of the gravid uterus, remained statistically significantly lower than control. In rats exposed to 1500 ppm of the test material, statistically significantly lower body weight gain was apparent during gestation days 17 to 20. Prior to this, no statistically significant difference in body weight gain had been apparent and there were no statistically significant differences from control animals apparent for cumulative body weight gain, including overall body weight gain when adjusted for the contribution of the gravid uterus. No statistically significant differences in body weight gain during gestation were apparent for females that received 500 ppm of the test material. Food consumption throughout gestation was statistically significantly lower in rats exposed to the test material at 3000 ppm when compared with control animals. No obvious effects on food consumption during gestation were observed in animals exposed to the test material at 500 or 1500 ppm.
Gross necropsy on Gestation Day 20, did not reveal any remarkable findings (macroscopic abnormalities) in adult animals in any of the treatment groups.
There was no effect of maternal dietary exposure 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 at dietary exposures of 500, 1500 or 3000 ppm of the test material.
Maternal dietary exposure to 3000 ppm of the test material resulted in statistically significantly lower mean foetal, litter and placental weights when compared to the control animals. At the 1500 ppm maternal dietary exposure level, mean foetal and placental weights were statistically significantly lower than the control group. Litter weight and total placental weight were also lower when compared to the control group, but differences failed to attain statistical significance. No statistically significant differences in mean foetal and placental weights were observed in the 500 ppm treatment group when compared to the control animals.
External examination at necropsy revealed a higher incidence of visually small foetuses in the 3000 ppm and, to a lesser extent, the 1500 ppm maternal dietary exposure groups, when compared to the control. No such effects were apparent in the 500 ppm treatment group. Neither the type, incidence nor the distribution of visceral findings observed in the foetuses indicated any effect of maternal dietary exposure to 500, 1500 or 3000 ppm of the test material.
Maternal dietary exposure to 3000 ppm of the test material, resulted in a clear effect on a large number of ossification parameters affecting most regions of the skeleton. There was (generally) an increase in both the number of foetuses and litters affected compared to the control group and statistically significant differences from control in foetal incidence for these parameters. At the 1500 ppm maternal dietary exposure level, there was a similar effect on ossification parameters, although changes were not quite as extensive and generally occurred at a lower incidence than that observed at the 3000 ppm dietary exposure level. Again, there was generally an increase in both the number of foetuses and litters affected compared to the control group and statistically significant differences from control in foetal incidence for these parameters. Neither the type, incidence nor the distribution of skeletal findings observed for foetuses indicated any effect of maternal dietary exposure to 500 ppm of the test item.
Based on the results of this study, dietary exposure to 500 ppm of the test material (equivalent to an achieved dosage of approximately 41.2 mg/kg bw/day of the test material) was considered to be a clear No Observed Effect Level (NOEL) for the pregnant dam. The No Observed Adverse Effect Level (NOAEL) for the pregnant dam was considered to be a dietary exposure to 1500 ppm of the test item (equivalent to an achieved dosage of approximately 122.9 mg/kg bw/day of the test material), due to effects on body weight gain and food consumption apparent at a dietary level of 3000 ppm.
The NOEL for the developing conceptus was considered to be a maternal dietary exposure to 500 ppm of the test item. The NOAEL for developmental toxicity was also considered to be at least 500 ppm of the test material, due to effects on foetal and placental weights and numerous skeletal findings indicating incomplete ossification or no ossification for many regions of the skeleton at the 1500 ppm level.
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