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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Nov 2014 - 07 April2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Adopted 22 January 2001)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Norbornene
- Physical state: solid at room temperature
- Analytical purity: >99.4% (GC method: 99.56%)
- Impurities:Toluol 0.23%(GC method: 0.23%)
- Purity test date: 22 October 2014
- Lot/batch No.: 0000122622
- Expiration date of the lot/batch: end of 2014
- Storage condition of test material: refrigerated (5°C) under argon atmosphere

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: approx. 200 g
- Fasting period before study: no data
- Housing: in groups of four animals in macrolon cages
- Diet (e.g. ad libitum): Altromin, No 1324 TPF, maintenance diet for rats and mice, breeding diet No. 1314 TPF, ad libitum
- Water: sterilised community tab water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Rel. humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Heat corn oil (measured amount) and test item to 60°C for 15 minutes. Pour test item into corn oil to produce the test solution, determine net weight and volume of the test item, calculate concentration (mg/mL) of the stock solution. Add required corn oil to produce the highest application dose for the 600 mg/kg dose level. Heat for one minute and vortex for 15 seconds. Then, produce two serial dilutions (1:1) for the mid and the low dose level, respectively. Store test materials at 5°C under argon until application.

DIET PREPARATION
n. a.

VEHICLE
- Justification for use and choice of vehicle: corn oil was used because of the low solubility of Norbornene in water
- Concentration in vehicle: 150/75/36.5 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Source: Sigma, catalogue No. C8267
- Purity: 100% (:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A validated GC/FID method was used for the determination of Norbornene in corn oil. Samples were diluted with i-hexane, and toluene was used as internal standard. 1 µL was injected split less. Instruments: column: ZB-624, ID 0.32 mm, 1.8µm; Temperature 40°C, 13 min isothermal,. Gas: hydrogen. Inlet: 250°C split less; Detector FID, 260°C; Flow 2 mL/min. The ratio of the areas test item/toluene was used for quantification. Retention times were 5.72 min for Norbornene and 9.21 min for toluene. Recoveries were in the range 103 - 112 % for solutions containing 37,5, 75, and 150 mg/mL. Linear calibration curve; y=0.00886x + 0.00731; r²= 0.99998. Further details: Appendix B
Details on mating procedure:
Animals were mated at the breeding facility; no details reported (section 3.1 of the study report)
Duration of treatment / exposure:
gestational days 6 through 20
Frequency of treatment:
daily
Duration of test:
24 days, including acclimatisation
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 300, and 600 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
Planned: 24/group
Successfully mated: 18/23/21/21 in controls/low/mid/high dose group, respectively
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: dose levels were selected based on the results as follows:
-OECD 422 combined repeated dose and reproduction toxicity study, oral gavage: minor effects but no clear toxicity pattern at the highest tested dose of 500 mg/kg bw.
-OECD 413 90-day inhalation study: treatment related changes in the ovaries of females exposed to 4.98 mg/m³. No effects at 2.02 gm³, i.e. the NOAEL was 582 mg/kg bw in this study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were not included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: days 3, 6, 9, 12, 15, 18, and 21

FOOD CONSUMPTION: Yes
- Food consumption for each animal group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: days 3, 6, 9, 12, 15, 18, and 21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: macroscopical examination following sacrifice. Uterine weight (not obtained from animals found dead during the study). Uterine content was examined for numbers of embryonic or foetal deaths, viable fetuses, and scars. In the ovaries, the number of corpora lutea was determined for pregnant animals.
Ovaries and uterine content:
The ovaries and uterine content were 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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Descriptive statistics: the arithmetic mean and the standard deviation was calculated for all grouped numerical data originating from monitoring the body weight, food consumption and gross pathology (fetal and placenta weights). Where appropriate, detailed column statistics were applied (minimum / maximum data, 25% quantiles, standard error, upper and lower confidence interval 95%).

Inductive statistics: for basic analysis the respective test item groups were compared to the vehicle group by assessing of statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level was set to 0.05. In case the basic analysis returned a statistical significance additional inductive statistical analysis was applied as outlined in the main decision tree (see appendix). Most statistical hypotheses in this study were best characterised as “many to one”– comparing a vehicle control vs. three treatment groups, respectively. Therefore the adequate analysis method was a one-way ANOVA (Analysis of variance), followed by a post hoc Dunnett´s t-test. Analysis of body weight data includes the influence of two different categorical independent variables – time and dose groups. Therefore, analysis by two-way ANOVA, followed by Bonferroni post-hoc test, was assumed as the most adequate analysis method for this set of data. For all calculations, the significance level was set to 0.05. These further inductive statistics were performed using Graph Pad Prism for Mac, Version 5. All statistical data are documented in the appendix of the study report.
Indices:
mean pre- and post-implantation loss
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs: salivation was seen in all treated groups. The incidence and severity increased with the dose. 2two rats were occasionally affected in the low dose group; in the mid dose group, single animals showed this sign, starting from day 9, and all animals were affected on day 20; at the high dose, 3 animals showed this signs on day 9, and almost all animals from day 11 onwards.
Mortalities: two mid dose rats dies immediately after dosing due to an application error. One high dose rat was found dead more than 8 hours after death, the cause of death could not be established.
Body weights: treatment had no effect on the gravid rat body weight of either test group
Food consumption: food consumption was comparable across all dose groups, i.e. no treatment-related effect was noted
Necropsy: a pale pancreas was noted in 19/20 high dose and in 2/20 mid dose dams. This observation was not noted in the vehicle and in the low dose groups. The macroscopic examination revealed no other pathological findings.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: developmental effects

Details on embryotoxic / teratogenic effects:
Uterus contents: the mean gravid uterus weight (with ovaries) was comparable across all groups.
Ovaries: the number of corpora lutea was comparable across all groups.
Implantations: the number was comparable across all groups.
Number of live fetuses: comparable across all groups.
Dead fetuses: no dead foetus seen in any group.
Early resorptions: mean number increased at the high dose level (0.8 versus 0.2 in the control), but not statistically significantly increased.
Pre- and post-implantation losses: minor differences lacking statistical significance. Two of the control animals showed an unusual high incidence of pre-implantation losses (10 and 12). Re-calculation without these two animals did also not reveal significant differences between the animal groups.
Foetal weight: weight at birth was significantly (p<0.001) reduced in male and female fetuses in all dose groups. The effect was dose-dependent and most prominent at the high dose [-11.4% (males and -11.8% (females)]. Birth weight reductions >10% are considered to be adverse.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean number of corpora lutea

 

Corpora lutea (n)

 

Left ovary

Right ovary

Total

Dose group

Mean

Mean

Mean

SD

600 mg/kg

6.9

6.6

13.5

1.7

300 mg/kg

6.8

6.6

13.3

1.7

150 mg/kg

6.3

6.9

11.1

2.4

Vehicle

6.5

6.7

13.2

1.9

 

Table 2: Mean number of implantations

 

Corpora lutea (n)

 

Left uterus horn

Right uterus horn

Total

Dose group

Mean

Mean

Mean

SD

600 mg/kg

6.1

6.2

12.3

1.6

300 mg/kg

5.7

6.2

11.9

1.6

150 mg/kg

5.7

6.1

11.8

1.2

Vehicle

5.5

5.3

10.8

3.1

 

Table 3: Mean number of foetuses and resorptions

 

Corpora lutea

Implantation sites

Live fetuses

Early resorptions

Scar

Dead fetuses

Dose group

Mean

Mean

Mean

Mean

Mean

Mean

600 mg/kg

13.5

12.3

11.5

0.8

0.0

0.0

300 mg/kg

13.3

11.9

11.5

0.2

0.0

0.0

150 mg/kg

11.1

11.8

11.5

0.2

0.0

0.0

Vehicle

13.2

10.8

10.2

0.2

0.0

0.0

Vehicle *

13.1

11.8

11.1

0.2

0.0

 0.0

 

Vehicle*: calculation that excludes two dams with unusually high number of pre-implantation losses (10 and 12, respectively)

Table 4: Indices of implantation losses

 

Pre-implantation loss (CL-IS)

 

Post-implantation loss (IS-LF)

Pre-implantation loss per group

Post-implantation loss per group

Dose group

Mean

Mean

Mean

Mean

600 mg/kg

1.2

0.8

9.76

7.05

300 mg/kg

1.4

0.4

11.76

3.73

150 mg/kg

1.3

0.3

11.02

2.64

Vehicle

2.4

0.7

22.22

6.55

Vehicle *

1.3

0.7

11.1

6.55

 

Legend:

Vehicle*: calculation that excludes two dams with unusually high number of pre-implantation losses (10 and 12, respectively)

CL = Corpora lutea
IS = Implantation site
LF = Life fetuses

Indices were calculated as follows:

Pre implantation loss: mean no. of corpora lutea – mean no. of implantations

Mean no. of resorptions: No. of resorptions / no. of litters

Mean pre implantation loss per group: (mean no. of corpora lutea – mean no. of implantations) * 100 /mean no. of implantations

Mean post implantation loss (%): (Total no. resorptions + total no. of dead fetuses) * 100 / no. of fetuses.

All calculations and statistical tests are documented within the appendix of the report.

 

Table 5: mean fetal birth weight and weight changes

 

 

Mean weight [g]

 

Both sexes

Males

Females

Weight change [%]

Versus controls

Dose group

Males

Females

600 mg/kg

4.76

4.91

4.65

-11.74

-11.8

300 mg/kg

5.01

5.11

4.90

-7.8

-7.0

150 mg/kg

506

5.19

4.94

-6.3

-6.3

Vehicle

5.38

5.54

5.27

 

 

 

Applicant's summary and conclusion

Conclusions:
The NOAEL of Norbornene in an OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group.
Executive summary:

The developmental toxicity and teratogenicity of Norbornene was studied in time-mated Wistar rats according to the OECD 414 test guideline and under GLP conditions. Norbornene, dissolved in corn oil, was administered by oral gave at dose levels of 0, 150, 300 and 600 mg/kg bw, based on the results of preceding studies [OECD 422 (oral) and 413 (inhalation)] which gave NOAEL values of 500 and 582 mg/kg bw, respectively.

 

The NOAEL of Norbornene in the OECD 414 study using rats was established at 300 mg/kg bw (gavage), based on highly significant foetal weight reduction at all tested dose levels (150, 300, and 600 mg/kg bw) that reached >10% in the high dose group. Maternal toxicity and teratogenicity were not seen in any dose group. A significant increase of supernummary ribs was seen in fetuses of the high dose group (40% versus 8% in the control) and on litter basis (90% vs. 39% in the vehicle control. Therefore, this finding is considered to be test-item related, but not teratogen.

Overall the NOAEL of Norbornene in the Wistar rat was 600 mg/kg bw for maternal toxicity and teratogenicity, and 300 mg/kg bw for developmental toxicity.

 

The study is considered valid and suitable for assessment.