Resin acids and Rosin acids, fumarated, esters with glycerol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-08-13 to 2015-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

deviations had no adverse impact on the scientific purpose of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, fumarated, esters with glycerol

EC Number:

307-051-0

EC Name:

Resin acids and Rosin acids, fumarated, esters with glycerol

Cas Number:

97489-11-7

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

Resin acids and Rosin acids, fumarated, esters with glycerol

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD®(SD) IGS BR strain

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: 171 to 275g
- Fasting period before study: not specified
- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): Ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage
- Acclimation period: not specified

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

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were preparedon four occasions during the course of the study and stored at room temperature
- Mixing appropriate amounts with (Type of food): A known amount of test item was mixed with a small amount of basal laboratory diet in a Robot Coupe Blixer 4 mixer until homogeneous. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further sixty minutes at a constant speed, setting 1 in a Hobart U200 mixer.
- Storage temperature of food: stored at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the dietary admixtures was previously determined by Envigo Research Limited, Shardlow, UK, Analytical Services (Harlan Study Number 41302579). Representative samples were taken of three of the admixtures and analyzed for concentration of RARA, fumarated, esters with glycerol (CAS 97489-11-7) at Envigo Research Limited., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 85 % to 105 % of the nominal concentration confirming the suitability and accuracy of the formulation procedure.

Details on mating procedure:

A total of ninety-six time-mated female Sprague-Dawley Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited,Margate, Kent. Animals were delivered intwo batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.

Duration of treatment / exposure:

gestation days 3 to 19 (inclusive)

Frequency of treatment:

continuously in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

Low (equivalent to 244.2 mg/Kg bw/day)

Doses / concentrations 3

Dose / conc.:

7 500 ppm

Remarks:

Intermediate (equivalent to 622.2 mg/Kg bw/day)

Doses / concentrations 4

Dose / conc.:

15 000 ppm

Remarks:

High (equivalent to 1164.7 mg/Kg bw/day)

No. of animals per sex per dose:

24/concentration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dose levels were selected in collaboration with the Sponsor Representative, based on available toxicity data including an OECD 422 Study in the rat (Harlan Study Number 41302579). In the OECD 422 Study, a dietary concentration of 15000 ppm was well tolerated during gestation, therefore dietary concentrations of 0 (Control), 3000, 7500 and 15000 ppm were selected for use in this pre-natal developmental study.
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded for each individual animal on gestation days 3, 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on gestation day 20. All animals were subjected to a full external and internal examination. The ovaries and uteri of pregnant females were removed and examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
i) Foetal sex
ii) External foetal appearance
iii) Foetal weight
iv) Placental weight

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [all per litter]

Statistics:

The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, an alternative multiple comparison test. All caesarean necropsy parameters and foetalparameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated
values using the Mann-Whitney ‘U’ test, where significance was seen. Foetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Indices:

Pre and Post Implantation Loss
i) Percentage pre-implantation loss was calculated as: (Number of corpora lutea - Number of implantations/Number of corpora lutea) x 100
ii) Percentage post-implantation loss was calculated as: (Number of implantations - Number of live foetuses/Number of implantations) x 100

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were detected.

One female in the 15000 ppm dietary exposure group had red staining around the ano-genital region on Day 11 of gestation. In isolation thiswas considered incidental and unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the 15000 ppm dietary exposure level, body weight gain was generally lower than control throughout gestation, with differences frequently attaining statistical significance and this lower weight gain was still apparent after values were adjusted for the contribution of the gravid uterus.

Body weight gain during gestation, including after adjustment for the contribution of the gravid uterus, was considered to be unaffected by dietary exposure to 3000 or 7500 ppm of the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At the 15000 ppm dietary exposure level, food consumption was generally lower than control between gestation days 3 and 11, with differences attaining statistical significance. A slight recovery in food consumption was evident thereafter.

Food consumption during gestation was unaffected by dietary exposure to 3000 or 7500 ppm

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected during the macroscopic examination of the pregnant females at termination on gestation day 20.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Details on maternal toxic effects:

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

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Basis for effect level:

other: Systemic Toxicity

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: effects on body weight and food consumption at the highest concentration tested.

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the 15000 ppm dietary exposure level, mean foetal weight for both sexes was lower than control, resulting in lower mean litter weights; differences from control attaining statistical significance.

At the 3000 and 7500 ppm dietary exposure levels, mean male foetal weights and mean foetal weights (both sexes combined) were statistically significantly lower than control. All individual mean litter values were within historical background control ranges (mean male foetal weight: 3.24-5.03g and mean foetal weight: 2.42-4.97g) and in isolation at these levels were considered to reflect normal biological variation.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Neither the type, incidence nor the distribution of findings observed during external examination of the foetuses at necropsy on gestation day 20 indicated any adverse effect of maternal dietary exposure on foetal development.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At the 15000 ppm exposure level, there was clear effect of treatment on a large number of ossification parameters affecting most regions of the skeleton, with the number of foetuses/litters increased compared with control and differences frequently attaining statistical significance. These parameters included incomplete ossification of the frontal, parietal, interparietal, occipital, squamosal, jugal, zygomatic process of maxilla and squamosal, premaxilla bones of the skull, no ossification of the frontal and presphenoid bones of the skull, incomplete ossification of the lumbar (neural) arch, incomplete/no ossification of the sacral (neural) arch, less than 4 caudal vertebrae ossified, no ossification of the sternebra, partially split xiphoid cartilage,wavy/thickened/incomplete ossification of the ribs, and no ossification/incomplete ossification of the pubis, and metacarpals. There was also a lower incidence of foetuses showing no ossification of the squamosal bone of the skull and ossification centre associated with 1st lumbar vertebra.

At the 3000 and 7500 ppm dietary exposure levels, the incidence and type of skeletal findings observed did not indicate any adverse effect of maternal treatment. The percentage incidence of some of the skeletal parameters did attain statistical significance versus control, but these differences were not considered to be indicative of adverse foetal effects.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Neither the type, incidence nor the distribution of findings observed during detailed visceral examination indicated any adverse effect of maternal dietary exposure on foetal development.

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

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: Developmental Toxicity

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: Skeletal findings: reduced ossification; wavy rib; delays in skeletal development

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

15 000 ppm

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 2. Group Mean Body Weight Change Values
Dietary Concentration (ppm)		Cumulative Body Weight Change (g) from Day 3 of Gestation
		4	5	8	11	14	17	20
0 (Control)	Mean	3.6	7.6	25.0	47.8	62.6	90.5	141.9
	SD	8.7	7.1	8.0	9.3	11.4	18.5	20.2
	n	24	24	24	24	24	24	24
3000	Mean	4.2	6.8	25.5	47.8	62.9	86.3	130.0
	SD	6.7	6.8	8.5	10.3	10.9	13.0	18.2
	n	24	24	24	24	24	24	24
7500	Mean	2.5	9.8	26.5	45.2	60.6	86.5	131.5
	SD	8.8	7.6	11.1	12.0	13.6	17.1	21.0
	n	24	24	24	24	24	24	24
15000	Mean	1.5	5.3	19.5	35.8**	51.4**	74.9**	116.3***
	SD	9.5	11.7	15.5	18.7	18.8	20.0	19.2
	n	24	24	24	24	24	24	24

Table 3. Group Mean Food Consumption Values
Dietary Concentration (ppm)		Food Consumption (g/rat/day) between Days of Gestation
		3-5	5-8	8-11	11-14	14-17	17-20
0 (Control)	Mean	18.0	21.8	24.8	23.7	26.8	27.0
	SD	3.7	3.4	3.2	3.4	3.7	3.4
	n	24	24	24	24	24	24
3000	Mean	17.1	21.7	24.2	24.9	25.6	26.0
	SD	4.4	3.3	2.9	3.0	2.8	3.7
	n	24	24	24	24	24	24
7500	Mean	18.7	21.3	23.3	24.4	26.8	26.9
	SD	3.3	2.4	2.1	2.3	3.3	3.1
	n	24	24	24	24	24	24
15000	Mean	15.1**	18.7**	20.1***	22.9	24.9	25.8
	SD	1.9	3.4	2.8	2.9	4.7	2.2
	n	24	24	24	24	24	24

Table 4. Group Mean Litter Data Values
Dietary Concentration (ppm)		Mean Male Foetal Weight (g)	Mean Female Foetal Weight (g)	Mean Foetal Weight (g)	Mean Placental Weight (g)	Litter Weight (g)	Total Placental Weight (g)
0 (Control)	Mean	4.173	3.961	4.067	0.576	54.353	7.675
	SD	0.228	0.265	0.237	0.064	10.790	1.624
	n	24	24	24	24	24	24
3000	Mean	3.986*	3.782	3.877*	0.553	49.742	7.110
	SD	0.295	0.313	0.289	0.067	8.359	1.464
	n	24	24	24	24	24	24
7500	Mean	3.956*	3.780	3.870*	0.571	49.805	7.233
	SD	0.243	0.246	0.233	0.109	10.162	1.490
	n	24	24	24	24	24	24
15000	Mean	3.636***	3.447***	3.545***	0.519	45.258***	6.658**
	SD	0.260	0.228	0.245	0.063	5.559	1.249
	n	24	24	24	24	24	24

Table 5. Summary Incidence of Foetal Skeletal Findings
Skeletal Findings	Dietary Concentration (ppm)
	0 (Control)			3000			7500				15000
	Number of Foetuses (litters) Examined
	155 (24)			148 (24)			148 (24)				148 (24)
	NF	NL	%†	NF	NL	%†		NF	NL	%†		NF	NL	%†
Skull
Frontal - incomplete ossification	7	4	4.7	4	4	3.0		14	7	9.3		31	14	20.1**
Frontal - unossified area	0	0	0.0	5	3	3.6		4	1	2.8		7	6	4.5*
Parietal - incomplete ossification	15	8	9.8	8	5	5.6		13	10	8.3		41	15	27.2*
Interparietal - incomplete ossification	31	13	20.1	29	12	20.4		35	12	23.3		74	22	49.8***
Occipital (Supra-occipital) - incomplete ossification	24	9	15.2	32	15	22.1		48	19	32.6**		73	21	48.9***
Squamosal - incomplete ossification	20	8	13.1	16	12	9.9		26	13	17.3		54	19	36.3**
Squamosal - unossified area(s)	8	6	5.7	1	1	0.7*		4	3	2.5		0	0	0.0*
Jugal - incomplete ossification	16	9	10.5	8	6	6.1		17	10	11.9		33	16	22.3*
Zygomatic process of maxilla - incomplete ossification	15	8	9.4	6	5	3.9		26	13	17.4		31	15	20.7*
Zygomatic process of squamosal - incomplete ossification	4	2	2.4	0	0	0.0		4	3	3.5		17	9	11.8*
Premaxilla - incomplete ossification	0	0	0.0	4	2	1.9		8	5	5.1		13	5	7.7*
Presphenoid - not ossified	1	1	0.5	1	1	0.5		0	0	0.0		11	7	6.9*
Vertebral Column
Lumbar (neural) arch - incomplete ossification	1	1	0.6	0	0	0.0		2	1	1.0		9	6	6.2*
Sacral (neural) arch - incomplete ossification	19	9	11.8	18	13	12.3		44	17	29.6		47	14	29.8*
Caudal vertebrae – less than 4 ossified	18	12	11.5	39	16	25.5		38	14	24.8		60	18	38.3**
Ribs
Ossification centre - associated with 1st lumbar vertebra	13	9	9.1	3	3	1.8*		8	7	5.4		3	2	1.9*
One or more ribs – wavy	2	1	1.4	4	2	3.8		5	4	4.0		27	10	17.9**
One or more ribs – thickened	2	2	1.4	4	3	3.5		6	5	4.6		23	9	15.7*
Rib - incomplete ossification	3	2	2.0	1	1	1.0		3	2	1.9		20	8	13.4*
Sternebrae
Sternebra - not ossified	0	0	0.0	0	0	0.0		6	1	3.1		7	5	4.3*
Xiphoid cartilage - partially split	9	5	7.9	20	11	15.7		9	7	6.7		23	15	14.9*
Pelvic Girdle
Pubis - not ossified	0	0	0.0	0	0	0.0		3	3	1.8		10	5	6.4*
Pubis - incomplete ossification	11	6	6.5	6	6	4.1		26	12	16.6		31	11	20.6*
Forelimb
Metacarpal - not ossified	18	10	12.1	40	18	27.4*		57	15	36.5*		81	20	52.7***
Metacarpal - incomplete ossification	3	3	2.3	1	1	0.8		10	6	6.7		22	11	14.4**

NF: Number of foetuses

NL: Number of litters

%†: Group mean percent

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

 

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

 

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

 

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

 

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

 

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

 

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

 

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