Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Dominant lethal assay: NOAEC (5 d, inhl): 254 ppm (1270mg/m3)

Effects on developmental toxicity

Description of key information
NOAEL (maternal toxicity, inhl): 25 ppm
NOAEL (developmental toxicity, inhl): 100 ppm
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline study. Well conducted and reported study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD (Sprague-Dawley)
Details on test animals and environmental conditions:
Each animal was assigned a unique number and was ear tagged for identification.

TEST ANIMALS
- Virgin male and female CD (Sprague Dawley).
- Age not recorded; acclimated for 2 weeks before exposure commenced.
- Weight at study initiation: females ~235 g.
- Housing: 2 (1M/!F) to a cage in stainless steel wire-mesh cages.
- Diet: Prolab Certified Rodent Food@, ad libitum except during exposure period
- Water: tapwater
- Acclimation period: two weeks during which time they were examined by the Clinical Veterinarian and representative animals were subjected to a full necropsy, examination for intestinal parasites, histologic examination of selected tissues and serum viral antibody analysis.

ENVIRONMENTAL CONDITIONS
- Temperature (exposure chamber): 20.4-22.2 Celsius
- Humidity (exposure chamber): 47.5-49.6%
- Photoperiod: 12-hour
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
For generation of atmospheres, liquid ENB was metered from a heated glass evaporator similar in design to that described by Carpenter et al. (1975) and, more recently, Snellings and Dodd (1990). The temperature in the evaporator(s) was maintained at the lowest level sufficient to vaporize the liquid (31-52°C). The resultant vapor was carried into the chamber by passage of conditioned air through the evaporator(s). Concentrations of the ENB vapor in the exposure chambers were monitored using a gas chromatograph equipped with a flame ionization detector.

Chamber volume: 3220litres, airflow 1000litres/min.

Chamber temperature, relative humidity, and airflow rate were recorded approximately every 30 minutes during each six-hour exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical chamber concentrations were measured by gas chromatography. Calibration of the gas chromatograph was done using a series of standards which encompassed the entire range of vapor concentrations generated in the exposure chambers. Each chamber atmosphere was monitored for concentration verification approximately once every 25 minutes during each daily six-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were calculated for each chamber. In a preliminary study on the distribution of vapur in the exposure chamber, each chamber was sampled at 5 different positions in triplicate. The variation seen was deemed acceptable.
Details on mating procedure:
- If cohoused:
- M/F ratio per cage: 1:1 (one male at least 300 g:one female at least 200 g) mating cages (22.5 x 31.0 x IS cm high)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Plug-positive females-were housed individually for the duration of the study. 
Duration of treatment / exposure:
Gestation days (GD) 6-15
Frequency of treatment:
6 hours/day
Remarks:
Doses / Concentrations:
0, 25, 100, or 354 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
25
Control animals:
other: yes, air-exposed only
Details on study design:
Sex: female
Duration of test: females sacrificed on GD21
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Maternal animals were observed daily for mortality, morbidity, and signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION : Yes, Maternal food consumption was measured at 3-day intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: general abdominopelvic gross pathology, gravid uterine weight, liver and kidney weights. Maternal thyroid glands were weighed and prepared for light microscopic examination and morphometric measurements.
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 resorptions: not reported
Fetal examinations:
All fetuses were weighed, sexed, and examined for external variations and malformations.
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal abnormalities ; these fetuses were decapitated and the heads fixed in Bouin's solution for examination of craniofacial structures by sectioning. 
- Skeletal examinations: Yes: All fetuses were eviscerated and processed for skeletal staining with alizarin red S to examine for skeletal malformations and variations.
- Head examinations: Yes: half per litter
Statistics:
Quantitative continuous variables were intercompared between ENB groups and the corresponding air-control groups using Levene's test for equal variances,analysis of variance, and t-tests. The t-test was used when the F value from the ANOVA was significant. Non-parametric data were statistically analyzed using the Kruskall-Wallis test followed by the Mann-Whitney U test. Incidence data were compared using Fisher's Exact Test. A probability value of <0.05(two-tailed) was used as the critical level of significance.
Dose descriptor:
NOAEL
Effect level:
25 ppm
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
.
Dose descriptor:
NOAEL
Effect level:
100 ppm
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

There were no maternal deaths or abortions. One dam at 100 ppm delivered early. Pregnancy rates ranged from 83.3 to 100% and were equivalent across all groups. Between 20 and 25 litters were available for examination in each group. There were no exposure-related clinical signs of toxicity. Gestational body weights were reduced GD 9, 12, 15, and 18 at 354 ppm and on GD 15 at 100 ppm. Gestational body weight gains were statistically significantly reduced (p< 0.01) 61.5% over GD 6-15 at 354 ppm and 25.2% over GD 9-15 at 100 ppm, both accompanied by decreased food consumption (21.4% and 10.4%, respectively, p< 0.01). At 100 ppm, food consumption was less markedly affected, being reduced 16.5% for GD 9-12 and 8.0% for GD 12-15. Food consumption was similar to controls in the post expsoure period.  

Necropsy: There were no effects on the number of corpora lutea. The corrected body weights and corrected body weight changes were reduced in the 354 ppm group. Liver weights showed a nonstatistical trend for increase at 100 and 354 ppm. Relative liver weight was increased at 100 and 354 ppm. There were no statistically significant differences among the groups for number of live fetuses per litter, percent malformations per litter, placental or fetal body weights, or sex ratio. Three skeletal variants were increased in litters of the 354 ppm group (bilobed 12th thoracic centrum, split 12th thoracic centrum, and poorly ossified second sternabra) and one variant (bilobed 12th thoracic centrum) was increased in the 100 ppm group litters, but these occurred in the presence of maternal toxicity and were not seen in the 25 ppm group. Whilst the incidence of bilobed 12th thoracic centrum was statistically increased relative to controls, there was no clear dose response relationship - incidence at all three doses was similar and the effect did occur in controls. The biological significance is therefore questionable in the absence of any other significant findings. Both absolute and relative thyroid weights were unaffected by maternal exposure to ENB. There was no difference in thyroid hormones compared to controls. Histology evaluation confirmed vacuolar colloid depletion in all groups, (including controls) that was more marked and numerous in ENB groups. Morphometric evaluation confirmed concentration-related depletion of thyroid colloid in the ENB groups. There were no clinical signs suggestive of thyroid dysfunction.

Executive summary:

Neeper-Bradley et al. (1995) reported results of a teratogenicity test of ENB conducted in CD (Sprague-Dawley) rats by inhalation of 0, 25, 100 and 354 ppm (0, 123, 492, 1740 mg/m3) during days 6-15 of pregnancy. There was no maternal mortality. Maternal body weights, body weight gain, and food consumption were reduced over the exposure period at 100 and 354 ppm, with partial or complete recovery post exposure. Increased relative liver weights were measured for the 100 and 354 ppm groups. Vacuolar depletion of thyroid follicular colloid was present in all groups of pregnant animals, including air-only controls, but the incidence and extent was greater in ENB vapor-exposed rats. There were no effects on tri-iodothyronine (T3), tetra-iodothyronine (T4) or T3 uptake. There were no increases in the incidence of malformations or external and visceral variations. Three skeletal variants (bilobed 12th thoracic centrum, split 12th thoracic centrum, and poorly ossified second sternabra) were increased at 354 ppm, and one (bilobed 12th thoracic centrum) was increased at 100 ppm. Thus, minimal developmental toxicity in the form of skeletal variants was observed in the 100 and 354 ppm group litters in the presence of maternal toxicity but analysis of the results suggests no dose response relationship and, in the absence of other findings, this change may not be biologically significant. On this basis, it is concluded that the NOAEL for maternal toxicity is 25ppm (123 mg/m3) whilst that for developmental toxicity/teratogenicity is 100 ppm (492 mg/m3)

Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Species:
rat
Quality of whole database:
Effects were only seen well above maternal toxicity threshold (125mg/m3), the latter being entirely consistent with the repeat dose toxicity information.
Additional information

A teratogenicity test of ENB was conducted in CD (Sprague-Dawley) rats by inhalation of 0, 25, 100 and 354 ppm (0, 123, 492, 1740 mg/m3) during days 6-15 of pregnancy (Neeper-Bradley, et al. 1995). A NOAEL of 25 ppm (123 mg/m3) was established for both maternal toxicity and a NOAEL of 100ppm (492 mg/m3) for developmental toxicity/teratogenicity.

Waiver for developmental toxicity study in second species: Whilst the regulation 1907/2006 is not clear on this, a study on a second species is often expected for a substance registered at >1000tpa. This substance is a monomer and chemical intermediate. There is only a single production site in Europe and a very limited number of industrial downstream user sites. The supply chain is very short, finishing with the polymer manufacturers and typically only involving one direct transaction from supplier or importer to the DU site. According to Annex XI of the regulation, the data requirements may be adapted under certain conditions. Specifically, paragraph 3.1 states that testing in accordance with Annex IX and X may be omitted based on exposure scenarios developed in the CSR. 1) The exposure assessments in the CSR covering all relevant exposures throughout the life cycle of the substance demonstrate that there is no significant exposure in all scenarios of the manufacture and all identified uses identified in chapter 3.5 of this dossier. 2) A DNEL can be derived from the results of the available data. Any uncertainty is resulting from the omission of this data is taken into account, although such uncertainty is reduced by the availability of screening data for this end point., and that the DNEL is relevant and appropriate both to this end point and for risk assessment purposes. 3) A comparison shows that the predicted exposures are well below the derived DNEL. In this case, because of the very low odour threshold of this substance and the need to control emissions to prevent odour release (very low odour threshold) and the fact that the substance is handled under strictly controlled conditions by all sites using >10tpa (see attached separate document in chapter 13 that describes the handling conditions). Taking all this evidence into account along with the requirements in the regulation not to carry out unnecessary animal tests, there does not appear to be a justifiable requirement for a developmental toxicity study in a second species.


Justification for selection of Effect on developmental toxicity: via inhalation route:
Only one study available.

Toxicity to reproduction: other studies

Additional information

In addition, a dominant lethal study produced no effect on the testes of rats exposed to ENB concentrations up to 254 ppm (1270mg/m3) for 5 days (Neeper-Bradley and Ballantyne, 1996). See study summary in Section 7.6.2.

Justification for classification or non-classification

Reliable animal data show no evidence of reproductive of developmental effects at levels below those producing toxicity, therefore, no classification is proposed.