Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in an established laboratory according to the Rodent Dominant Lethal Assay Guideleines of the U.S. EPA (1982) and in full compliance with GLP.

Data source

Reference
Reference Type:
publication
Title:
Absence of dominant lethal effects in male CD® rats exposed to 5-ethylidene-2-norbornene vapor.
Author:
Neeper-Bradley, T. and Ballantyne, B.
Year:
1996
Bibliographic source:
Toxic Substance Mechanisms 15, 389-404.

Materials and methods

Principles of method if other than guideline:
This study design was based on guidelines supplied by the United States EPA for the "Rodent Dominant Lethal Assay" (1982). Other sources examined for development of the study design included Green et al (1976; 1985) and Ehling et a1. (1978).
GLP compliance:
yes
Type of assay:
rodent dominant lethal assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance: 5-Ethylidene-2-norbornene (CAS No. 16219-75-3)
Reference no 1-SCD-113.
Purity: 99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
All animals were assigned a unique number and identified by cage tags.

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Age at study initiation: 70 days of age
- Housing: During the acclimation period and prior to mating, animals were housed separated by sex, 1 male or 2 females to a cage, in stainless steel wire-mesh cages. After the exposure period, the males were housed in stainless steel wire-mesh cages with 2 females weekly, for mating. After successful mating, the females were housed individually.
- Diet: Ground, certified Rodent Diet (RMH 3200, Agway, Inc.) was available ad libitum, except during exposures.
- Water: Tap water was available ad libitum, except during exposures.
- Acclimation period: The acclimation period for each shipment of animals was approximately 2 weeks. During this period, the animals were weighed at least 2 times at scheduled intervals.

ENVIRONMENTAL CONDITIONS
- Temperature: 66-75°F.
- Humidity: 40-70%
- Photoperiod: 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase).

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
No
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure cages were stainless steel with glass windows for observation and were approximately 1330 liters
- System of generating particulates/aerosols: Liquid ENB was metered from a piston or syringe pump into a heated glass evaporator (55-66 Celsius) similar in design to that described by Snellings and Dodd (1990). The resulting ENB vapor was carried into the exposure chamber by a countercurrent air stream that entered the bottom of the evaporator and flowed into the chamber from the top of the evaporator.
- Temperature: 21.8-22.6 Celsius.
- Humidity: 55.1-61.4%,
- Air flow rate: approximately 300 liters/minute
- Air change rate: 13 air changes each hour
Duration of treatment / exposure:
6 hours/day for 5 days
Frequency of treatment:
5 consecutive days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 52, and 254 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
20 exposed males per group
40 untreated females per group were used for each mating week
Control animals:
other: Yes, exposed to filtered air. Following the final exposure to filtered air, positive control males received a single intraperitoneal injection of 0.5 mg TEM/kg body weight.
Positive control(s):
Triethylenemelamine

Examinations

Evaluation criteria:
Reproductive/gestational parameters examined included: No. impregnated females/no. females paired (mating index, females):
No. impregnating males/no. males paired (mating index, males):
No. pregnant females/no. females impregnated (fertility index, females):
No. males producing pregnant females/no. males impregnating (fertility index, males):
No. corpora lutea/pregnant female:
No. total implantations/pregnant female:
Preimplantation loss/pregnant female (%):
No. nonviable implantations (early and late resorptions and
dead fetuses)/pregnant female:
Postimplantation loss/pregnant female (%):
No. females with 1 or more dead implantations (%):
No. females with 2 or more dead implantations (%):
Ratio of nonviable implantations/total implantations:
FL% (the dominant lethal factor) =[ 1 - live implantations/female of test group ] X 100 / live implantations/female of control group
Statistics:
The unit of comparison was the male or the pregnant female (Weil, 1970). Results of the quantitative continuous variables (for example, male body weights) were intercompared for the 3 ENB-treated groups and vehicle control group by use of Levene's test for equal variances (Levene, 1960), analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1970) followed, when necessary, by the separate variance t-test. The positive control group was compared to the vehicle control group using the previously described set of statistics.
Nonparametric data were examined statistically using the Kruskal-Wallis test (Sokal and Rohlf, 1969) followed by the Mann-Whitney U test (Sokal and Rohlf, 1969) when appropriate. Frequency data were compared using the Fisher's Exact Test (Sokal and Rohlf, 1969). For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight transient body weight effects at high dose
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

ENB did not produce evidence of dominant lethality in rats using a standard protocol (Neeper-Bradley and Ballantyne, 1996). Male rats exposed to analytically measured ENB vapor concentrations of 0, 5, 52, or 254 ppm for 6 hr/day for 5 consecutive days were mated with unexposed females.

Reproductive factors, including the number of fertile males and the number of gravid females with viable implants, were unaffected by exposure to ENB vapor. No significant preimplantation loss or dominant lethal effects were observed in females mated to ENB-exposed males during the 10-week breeding period. Males exposed to 254 ppm had slightly reduced body weight gains after the 5-day treatment period, but body weights and body weight gains were comparable to the controls thereafter. Triethylenemelamine (0.5 mg/kg, single dose on day 5 as a positive control) produced clear dominant lethal effects.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The ENB-exposed males showed no clinical signs of toxicity. The only objective finding was slight decrease in body weight gain at 254 ppm over the exposure period, but which resolved by the end of the first postexposure week. No reproductive or gestational effects, including dominant lethality, were noted in the ENB-treated groups during the 10 wk of postexposure mating. This finding confirms the absence of testicular toxicity in the rat from repeated exposure to high concentrations of ENB vapor.
Executive summary:

Neeper-Bradley and Ballantyne (1996) reported that ENB did not induce dominant lethal mutations in rats exposed to concentrations up to 254 ppm for 5 consecutive days (6 hr/day). The NOEC for toxicity (body weight gain) was 52 ppm. The NOEC for dominant lethality was greater than 254 ppm.