Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information

Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol was tested in an Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat according to OECD 414.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 25 August 2016 and 04 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147
Version / remarks:
24 November 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification : Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol
Physical State/Appearance : Beige solid flakes
Chemical Name : Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol
CAS Number : 68183-39-1
Trade Name : Aradur 3380-1 CH
Purity : UVCB Substance; purity not applicable
Batch Number : AAE1455700
Label : Aradur 3380-1 CH Benzene-1,2,4-tricarboxylic acid 1,2- anhydride, oligomeric reaction products with ethane-1,2- diol and glycerol Batch Number AAE1455700 Expire Date 10.10.2018 Net 1L
Date Received : 15 July 2016
Storage Conditions : In the dark at room temperature
Expiry Date : 10 October 2018

No correction for purity was made.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD® (SD) IGS BR strain
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in three batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 195 to 316g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex 2. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; there were short term deviations from the humidity target ranges which were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
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.
Route of administration:
oral: gavage
Details on exposure:
The test item was prepared at the appropriate concentrations as a suspension in arachis oil.

Dose Administration
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Control 0 4 0 24 (1-24)
Low 100 4 25 24 (25-48)
Intermediate 300 4 75 24 (49-72)
High 1000 4 250 24 (73-96)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.
The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

3.2.3 Preparation of Calibration Standards
Stock solutions of test item in dilution solvent (see Section 2.2.2) were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section 2.2.6.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.106 mg/mL by serial dilution covering the concentration range 0.0553 mg/mL to 0.2212 mg/mL.

Preparation of Test Samples
The formulations received were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extract solvent this was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration. See Table 1 for preparation details.
Preparation of Accuracy and Precision Samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations.
The concentration of Test Item in the final solution was quantified by HPLC using UV detection as detailed in the instrument parameters section.

Instrumentation Parameters
HPLC: Agilent Technologies 1100 or 1200, incorporating autosampler and workstation
Column: Luna 5µ C18 (250 x 4.6 mm id)
Mobile phase: Acetonitrile
Flow-rate 1 mL/min
UV detector wavelength: 250 nm
Injection volume: 25 µL
Retention time: ~ 2.9 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concentrations of 2.5 mg/mL and 250 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and Stability in Vehicle Formulations
The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed at nominal concentrations of 2.5 mg/mL and 250 mg/mL (homogeneity), 25 mg/mL and 250 mg/mL (stability) during ambient storage.

Stability
The formulations were analyzed on receipt. After 4 hours; the formulations were mixed as stated in the mixing procedure (described in the main part of this study) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for Test Item in Arachis Oil BP with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Results are summarized below.
The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The data was found to have a linear correlation within the calibration range of 0.0553 mg/mL to 0.2212 mg/mL. The R² fit of the calibration curve to the data was 0.9977, and was considered to be acceptable.
A mean recovery value of 99% (CV=2.23%, n=5) was obtained for 2.5 mg/mL and 96% (CV=3.46%, n=5) was obtained for 250 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed with respect to the level of concentration at nominal concentrations of 2.5 mg/mL and 250 mg/mL (homogeneity), 25 mg/mL and 250 mg/mL (stability).

Concentration of Dose Formulations
The mean concentrations of Test Item in test formulations analyzed during the study indicated that the mean concentrations were within applied limits ±12%, confirming accurate formulation (with the exception of analysis 3).

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for Test Item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL(homogeneity), 25 mg/mL and 250 mg/mL (stability).when stored for 4 hours.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±12% of nominal concentrations, confirming accurate formulation (with the exception of analysis 3).






Details on mating procedure:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in three batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 195 to 316g.
Duration of treatment / exposure:
The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Once daily
Duration of test:
20 Days
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.
Clinical signs, body weight change, food and water consumptions were monitored during the study.
All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
Individual body weights were recorded on Day 3 (start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each surviving individual animal on Day 3, 5, 8, 11, 14, 17 and 20 of gestation

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

POST-MORTEM EXAMINATIONS: Yes
Necropsy
All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight vii) Gravid uterus weight

Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position.
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage
OTHER:
Ovaries and uterine content:
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) Fetal sex
iv) External fetal appearance
v) Fetal weight vi) Placental weight
vii) Gravid uterus weight
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position
Fetal examinations:
The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
3
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 fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal 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
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 fetuses) / number of implantations x 100

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = Number of male fetuses / Total number of fetuses x 100

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day the majority of females showed both an increase in noisy respiration and increased post dose salivation from Gestation Day 7 onwards. There were sporadic instances when females showed respiratory observations, hunched posture and pilo-erection during the treatment period.
At 300 and 100 mg/kg bw/day females also exhibited clinical signs of increased salivation and noisy respiration.
The respiratory observations, in particular noisy respiration, showed a dose related response suggesting the test item may be irritant in nature. Therefore these observations were considered not to be of toxicological significance in terms of systemic effects.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female 83 treated with 1000 mg/kg bw/day showed clinical signs of a mass under the right forelimb which suggests trauma caused by the dosing procedure, hypothermia, labored respiration, decreased respiratory rate, hunched posture, pilo-erection and staining of the snout. There were no obvious effects on body weight or food consumption. The animal was killed in extremis on Day 9 of gestation and the macroscopic examination revealed enlarged adrenal glands and the mass contained pale yellow fluid and brown granular solids. This premature death is considered to be the result of dosing trauma and not test item related.
Female 89 treated with 1000 mg/kg bw/day showed body weight loss of 45g prior to death on Day 8 of gestation. There were clinical signs of gasping respiration, decreased and labored respiration, hunched posture, pilo-erection and stained fur. There were no obvious effects on food consumption. The macroscopic examination revealed gaseous distention in the stomach, duodenum, jejunum, ileum and cecum. These observations suggest that death may have resulted from the aspiration of the test item at dose administration.
There were no further unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day body weight gains were generally similar to controls during the first week of treatment, attaining a statistically significant increase between Days 4 and 5. Group mean body weight gains were slightly lower between Days 5 and 8 in relation to controls as three females showed actual body weight losses. Improvement was noted during the second week of treatment, but the animals showed lower weight gains during the final week of treatment attaining statistically significant lower body weight gains between Days 17 and 20. These body weight changes were reflected in the cumulative body weight gains.
When overall body weight gains were adjusted for gravid uterus weight, the body weight gains remained lower than controls without attaining statistical significance.
At 300 or 100 mg/kg bw/day body weight gains and cumulative gains were generally similar to controls with females treated with 300 mg/kg bw/day attaining statistical significance on Days 4 to 5 and Days 3 to 5 for body weight gains and cumulative body weight gains, respectively. Adjusted body weight gains were also generally similar to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day food intake was marginally lower than controls over Days 11 to 20, achieving statistical significance between Days 17 and 20. The food intake at 300 or 100 mg/kg bw/day was generally similar to controls.
Food efficiency:
not examined
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
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic abnormalities detected in the surviving females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Description (incidence and severity):
See "Any other information on results incl. Tables"
Description (incidence and severity):
See "Any other information on results incl. Tables"
Description (incidence and severity):
See "Any other information on results incl. Tables"
Early or late resorptions:
not specified
Description (incidence and severity):
See "Any other information on results incl. Tables"
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment related effects detected
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Description (incidence and severity):
See "Any other information on results incl. Tables"
Description (incidence and severity):
See "Any other information on results incl. Tables"
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
See "Any other information on results incl. Tables"
Skeletal malformations:
no effects observed
Description (incidence and severity):
See "Any other information on results incl. Tables"
Visceral malformations:
no effects observed
Description (incidence and severity):
See "Any other information on results incl. Tables"
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Table 1 Group Mean Litter Data Values

Dose Level

(mg/kg bw/day)

 

Number of

Corpora Lutea

Number of

Implants

Number of

Embryonic/Fetal Deaths

Implantation

Loss 

%

Number of Live Implants

%

Male

Fetuses

Mean

Male

Fetal

Weight

(g)

Mean

Female Fetal

Weight

(g)

Mean

Fetal

Weight

(g)

Mean

Placental

Weight

(g)

Litter

Weight

(g)

Total

Placental

Weight

(g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (Control)

mean

sd

15.2

1.5

14.5

1.5

0.2

0.5

0.2

0.6

0.4

0.9

4.4

5.8

2.8 7.0

7.3

1.9

6.7

2.3

14.0

1.9

52.5

12.9

4.159

0.292

3.943

0.253

4.067

0.269

0.574

0.056

56.843

7.718

8.032

1.341

 

n

23

23

24

24

24

23

23

24

24

24

24

24

24

24

24

24

24

100

mean

sd

15.9

2.5

14.6

1.8

0.1

0.4

0.4 0.8

0.5 0.9

7.4

7.9

3.7 6.4

7.1 2.1

7.0

2.1

14.1

1.9

50.4

13.6

4.198 0.234

3.986

0.221

4.093 0.220

0.563

0.073

57.505

7.290

7.870

1.114

 

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

300

mean

sd

16.2

3.0

15.1

2.4

0.1

0.4

0.3 0.6

0.4 0.7

6.0

7.9

2.8 4.8

7.0 2.4

7.7

1.9

14.7

2.5

47.3

12.5

4.106 0.288

3.913

0.294

4.004 0.281

0.558

0.059

58.580

9.706

8.200

1.646

 

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

1000

mean

sd

16.2

2.8

14.0

3.6

0.1

0.5

0.4

0.7

0.5

0.9

13.1

18.8

3.1

5.8

6.7

2.7

6.9

2.2

13.5

3.4

48.7

12.6

4.066

0.383

3.863

0.345

3.959

0.365

0.570

0.084

53.233

13.020

7.590

1.931

 

n

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

22

Fetal Examination

There were no treatment-related effects detected on external fetal examination findings and there were no treatment-related effects detected in the type and incidence of skeletal or visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

There was a statistically significant decrease in incidence of incomplete ossification of the parietal bone for 100 mg/kg bw/day litters and incomplete ossification of the humerus for 300 mg/kg bw/day litters. These findings are considered not to be of toxicological significance on the basis of no dose response relationship and because reduced incidences of incomplete ossification of individual bones generally implies better skeletal development.

Table 2 Summary Incidence of Fetal External Findings

 

External Findings

 

Dose level (mg/kg bw/day)

 

 

 

0 (Control)

100

300

 

1000

 

 

Number of fetuses (litters) examined

 

 

 

337 (24)

338 (24)

352 (24)

 

298 (22)

 

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Total Number Affected

 

7

5

2.1

2

2

0.6

7

6

2.0

3

2

0.8

Hematoma present

 

2

2

0.6

2

2

0.6

4

3

1.2

0

0

0.0

Small

 

1

1

0.3

0

0

0.0

2

2

0.6

3

2

0.8

Pale

 

4

2

1.2

0

0

0.0

1

1

0.3

1

1

0.3

Table 3 Summary Incidence of Fetal Visceral Findings

Visceral Findings

 

 

 

 

Dose Level (mg/kg bw/day)

 

 

 

 

 

0 (Control

)

 

100

300

 

 

1000

 

 

 

 

 

Number of Fetuses (litters) Examined

 

 

 

 

 

172 (24)

 

 

176 (24)

180 (24)

 

 

156 (22)

 

NF

 

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Head

Rugae - non-uniform patterning

 

12

 

 

9

 

7.5

 

12

 

10

 

7.0

 

10

 

9

 

6.3

 

17

 

10

 

11.9

Microphthalmia

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Abdomen

Liver - additional lobe between right and left median

 

2

 

 

2

 

1.2

 

0

 

0

 

0.0

 

0

 

0

 

0.0

 

0

 

0

 

0.0

Liver - papillary process - reduced in size

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Spleen - reduced in size

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Umbilical artery - left-sided

2

 

2

1.2

0

0

0.0

1

1

0.7

3

3

1.6

Ureter - kinked

12

 

8

7.8

10

8

5.5

4

3

2.2

8

6

6.1

Ureter - dilated - Slight/Severe

7

 

5

4.6

6

6

3.5

4

3

2.2

5

4

3.7

Kidney - reduced in size

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Renal pelvic cavitation - increased -

Slight/Severe

10

 

6

6.2

10

6

5.4

5

3

3.0

7

3

5.3

Renal papilla - absent

3

 

2

2.0

2

2

1.1

0

0

0.0

1

1

0.6

Renal medulla - reduced in size

1

 

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Thorax

Thymus - lobe partially undescended

 

6

 

 

4

 

3.4

 

5

 

5

 

2.6

 

0

 

0

 

0.0

 

8

 

5

 

4.5

Lungs - irregular surface throughout

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Atrium - enlarged

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Ventricular wall - thickened

0

 

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Total

36

 

19

22.3

28

18

15.6

19

13

11.5

35

15

23.0

Table 4 Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

 

0 (Control)

100

300

 

1000

 

 

Number of Fetuses (litters) Examined

 

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

 

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL

Fontanelle (anterior) - large

 

1

 

1

 

0.6

 

0

 

0

 

0.0

 

0

 

0

 

0.0

 

2

 

1

 

1.3

Nasal - incomplete ossification

6

5

3.6

4

3

2.3

13

6

6.6

3

3

2.1

Frontal - incomplete ossification

5

5

3.0

0

0

0.0

4

1

1.6

1

1

0.8

Frontal - unossified area

1

1

0.6

1

1

0.6

2

2

1.2

1

1

0.6

Parietal - incomplete ossification

9

7

5.8

0

0

0.0*

7

4

3.4

2

2

1.4

Parietal - unossified area(s)

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

Parietal/Interparietal - sutural bone

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.8

Interparietal - incomplete ossification

23

11

13.9

11

9

6.6

23

11

11.5

7

5

5.6

Interparietal - unossified area(s)

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Occipital (Supra-occipital) - incomplete ossification

12

8

7.1

11

6

6.8

14

8

7.1

15

7

10.8

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

6

5

3.3

4

2

2.6

6

6

3.5

8

5

5.2

Occipital - bipartite ossification

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Table 4 (Continued) Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

100

300

 

1000

 

Number of Fetuses (litters) Examined

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

SKULL (continued)

Squamosal - incomplete ossification

 

13

 

11

 

7.6

 

7

 

6

 

4.2

 

10

 

5

 

4.7

 

9

 

8

 

8.5

Squamosal - unossified area(s)

2

2

1.3

4

4

2.2

1

1

0.6

3

3

1.8

Jugal - incomplete ossification

4

3

2.4

0

0

0.0

4

3

2.0

1

1

0.8

Zygomatic process of maxilla - incomplete ossification

10

7

6.1

5

4

2.9

21

9

11.3

5

4

4.2

Zygomatic process of squamosal - incomplete ossification

1

1

0.6

0

0

0.0

1

1

0.6

1

1

0.8

Hyoid - incomplete ossification

12

7

7.7

11

9

6.6

10

10

5.7

4

4

2.4

Hyoid - not ossified

13

5

7.7

12

9

7.0

10

7

5.6

5

4

3.5

Tympanic annulus - incomplete ossification

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Presphenoid - incomplete ossification

2

1

1.2

0

0

0.0

2

1

0.7

2

1

1.5

Basisphenoid - incomplete ossification

0

0

0.0

1

1

0.7

0

0

0.0

1

1

0.8

VERTEBRAL COLUMN

Odontoid - ossification present

 

1

 

1

 

0.6

 

2

 

2

 

1.3

 

2

 

2

 

1.3

 

0

 

0

 

0.0

Ventral arch of vertebra 1 - ossification present

55

19

34.3

56

19

34.6

49

20

29.3

45

16

33.2

Cervical (neural) arch - incomplete ossification

2

2

1.2

4

4

2.5

7

4

3.4

6

4

3.8

Cervical (neural) arch - reduced in size

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

Cervical (neural) arch - misshapen

2

2

1.1

0

0

0.0

0

0

0.0

0

0

0.0

Cervical (neural) arch - fused

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Table 4 (Continued)Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

100

300

 

1000

 

Number of Fetuses (litters) Examined

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

VERTEBRAL COLUMN (continued)

Thoracic centrum - incomplete ossification

 

1

 

1

 

0.5

 

2

 

2

 

1.2

 

4

 

4

 

2.2

 

6

 

5

 

3.8

Thoracic centrum - bipartite ossification

5

5

2.9

0

0

0.0

1

1

0.6

1

1

0.9

Thoracic centrum - dumb-bell-shaped

11

9

6.7

12

8

7.9

8

5

4.6

12

8

10.0

Thoracic centrum - asymmetrically ossified

3

2

1.8

1

1

0.7

2

2

1.2

2

2

2.4

Thoracic (neural) arch - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Lumbar centrum - bipartite ossification

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Lumbar centrum - dumb-bell-shaped

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Lumbar centrum - asymmetrically ossified

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Sacral centrum - not ossified

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Sacral centrum - incomplete ossification

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Sacral (neural) arch - incomplete ossification

14

10

8.4

7

7

4.4

14

6

6.9

10

6

7.8

Sacral (neural) arch - not ossified

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Caudal vertebrae - less than 4 ossified

26

12

15.6

21

11

13.4

18

10

10.1

38

13

26.5

Caudal vertebrae - more than 6 ossified

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Number of pre-sacral vertebrae = 25/27 

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Scoliosis

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Table 4 (Continued)Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

100

300

 

1000

 

Number of Fetuses (litters) Examined

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

RIBS

Ossification centre - associated with 7th cervical vertebra

 

0

 

0

 

0.0

 

2

 

2

 

1.2

 

0

 

0

 

0.0

 

2

 

2

 

1.1

Ossification centre - associated with 1st lumbar vertebra

5

5

3.5

4

3

2.7

8

4

5.3

3

3

2.1

One or more ribs - wavy

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

One or more ribs - thickened

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

Rib - short

1

1

0.5

0

0

0.0

0

0

0.0

1

1

0.6

Rib - rudimentary

1

1

0.6

0

0

0.0

2

2

1.2

0

0

0.0

Costal cartilage - misaligned

2

2

1.2

6

6

3.9

0

0

0.0

6

5

4.0

Costal cartilage - not fused to sternebra

9

8

5.4

12

10

7.6

11

8

7.2

12

8

9.9

STERNEBRAE

Sternebra - incomplete ossification

 

1

 

1

 

0.6

 

0

 

0

 

0.0

 

1

 

1

 

0.3

 

4

 

4

 

2.6

Sternebra - not ossified

1

1

0.6

0

0

0.0

1

1

0.6

1

1

0.6

Sternebra - bipartite ossification

2

2

1.2

3

3

2.0

0

0

0.0

1

1

0.8

Sternebra - misaligned

4

3

2.0

7

6

4.5

3

3

1.5

7

6

4.9

Sternebra – fused

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Xiphoid cartilage - partially split

8

7

5.2

15

10

9.4

12

9

7.1

4

4

2.8

Table 4 (Continued)Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

100

300

 

1000

 

Number of Fetuses (litters) Examined

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

PECTORAL GIRDLE

Scapula - misshapen (comment on region)

 

5

 

3

 

2.6

 

2

 

2

 

1.3

 

9

 

5

 

5.2

 

4

 

4

 

3.6

PELVIC GIRDLE

Ischium - incomplete ossification

 

2

 

2

 

1.2

 

0

 

0

 

0.0

 

3

 

1

 

1.3

 

0

 

0

 

0.0

Pubis - not ossified

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Pubis - incomplete ossification

8

5

4.8

2

1

1.2

7

4

3.5

4

4

2.4

FORELIMBS

Metacarpal - not ossified

 

19

 

11

 

11.3

 

11

 

6

 

7.1

 

27

 

11

 

15.1

 

20

 

11

 

15.0

Metacarpal - incomplete ossification

3

3

1.8

0

0

0.0

2

1

0.7

0

0

0.0

Forepaw phalanges - 1 or more - ossified

31

12

20.0

40

14

23.5

51

19

30.7

51

17

36.8

Humerus - incomplete ossification

3

3

1.8

1

1

0.6

0

0

0.0*

0

0

0.0

Humerus - hole

0

0

0.0

1

1

0.8

1

1

0.5

2

2

1.3

Table 4 (Continued)Summary Incidence of Fetal Skeletal Findings

Skeletal Findings

 

Dose Level (mg/kg bw/day)

 

 

0 (Control)

100

300

 

1000

 

Number of Fetuses (litters) Examined

 

 

165 (24)

162 (24)

172 (24)

 

142 (22)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

HINDLIMBS

Metatarsal - 1st - ossified

 

5

 

2

 

3.4

 

1

 

1

 

0.7

 

8

 

3

 

5.0

 

0

 

0

 

0.0

Metatarsal - not ossified

1

1

0.6

0

0

0.0

1

1

0.6

1

1

0.6

Metatarsal - incomplete ossification

0

0

0.0

0

0

0.0

0

0

0.0

3

2

1.9

Hindpaw phalanges - 1 or more ossified

0

0

0.0

0

0

0.0

1

1

0.7

0

0

0.0

Femur - incomplete ossification

4

3

2.4

2

2

1.4

2

2

1.5

5

3

5.0

Femur - bent

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Fibula - bent

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

Total

135

24

81.9

125

24

76.4

153

24

89.2

123

22

87.7

Conclusions:
Based on the results of this study the No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) for the pregnant females was considered to be 1000 and 300 mg/kg bw/day, respectively. A dosage of 1000 mg/kg bw/day was considered to be the No Observed Effect Level (NOEL) for developmental toxicity
Executive summary:

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

      US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

• Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

• OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

• Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day.  A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.  

Clinical signs, body weight change, food and water consumptions were monitored during the study.  

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents.  The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded.  Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

Two females (No. 83 and 89) treated with 1000 mg/kg bw/day were terminated prematurely due to clinical signs on Gestation Days 9 and 8, respectively.  These deaths were considered to be the result of dosing trauma and not test item related.  

There were no further unscheduled deaths.

Clinical Observations

At 1000 mg/kg bw/day the majority of females showed increased salivation and noisy respiration and at a lesser extent at 300 or 100 mg/kg bw/day.  There were isolated incidences of females at 1000 mg/kg bw/day showing signs of laboured respiration, decreased respiratory rate and gasping respiration.

Body Weight

At 1000 mg/kg bw/day, females showed slightly lower mean body weight gains than controls towards the end of the gestation (Days 14 to 17 and 17 to 20) which resulted in statistically significantly lower overall body weight gain for these animals.  When overall body weight gain for this group was adjusted for the gravid uterus weight, the body weight gains remained lower than controls but without achieving statistical significance.  No such effects were detected for females treated with 300 or 100 mg/kg bw/day.  

Food Consumption

At 1000 mg/kg bw/day, there was an indication of marginally lower food intake from Day 11, achieving statistical significance between Days 17 and 20.  The food intake at 300 or 100 mg/kg bw/day was generally similar to controls.

Water Consumption

No adverse effect on water consumption was detected.  

Post Mortem Studies

No macroscopic abnormalities detected in the surviving females.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Fetal Examination

No treatment-related effects were detected on external fetal examination findings.  No treatment-related effects were detected in the type and incidence of skeletal or visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

Conclusion

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) for the pregnant females was considered to be 1000 and 300 mg/kg bw/day, respectively.  A dosage of 1000 mg/kg bw/day was considered to be the No Observed Effect Level (NOEL) for developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 GLP conform study performed according to OECD Guideline.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the above assement on oral repeated dose toxicity, Benzene-1,2,4-tricarboxylic acid 1,2-anhydride, oligomeric reaction products with ethane-1,2-diol and glycerol does not need to be classified for reprotoxicity according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.