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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
2 (reliable with restrictions)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Crl:CD1BR rats were selected for use on the two-generation reproduction study. Males and females (no male-female siblings) assigned to the reproduction study were approximately 8 and 11 weeks old, respectively, at the start of the study. During the cohabitation period, rats were housed as breeding pairs in stainless steel, wire-mesh cages. Females without evidence of copulation by the end of the cohabitation and assumed-pregnant females from gestation day 14 through delivery, were housed individually in polycarbonate pans with bedding. During the lactation period, adult females were housed with their litters in polycarbonate pans with bedding. The animals were exposed whole-body to atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane. The atmospheric concentration of cyclohexane was determined by gas chromatography at approximately 15-minute intervals during each 6-hour exposure. Prior to mating, P0 and P1 parental animals were exposed whole-body to the test substance for 6 hours/day, 5 days/week, excluding holidays. Pregnant females were exposed daily during days 0-20 of gestation.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexanone
EC Number:
203-631-1
EC Name:
Cyclohexanone
Cas Number:
108-94-1
Molecular formula:
C6H10O
IUPAC Name:
cyclohexanone
Details on test material:
Analytical purity: > 99%.

Test animals

Species:
rat
Strain:
other: Crl:CD1BR rat
Sex:
male/female

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: nitrogen and air
Doses / concentrationsopen allclose all
Dose / conc.:
7 000 ppm
Dose / conc.:
2 000 ppm
Dose / conc.:
500 ppm
Dose / conc.:
0 ppm
No. of animals per sex per dose:
30/sex/concentration level
Control animals:
yes

Examinations

Parental animals: Observations and examinations:
All adult P0 and P1 rats were weighed weekly during the premating, gestation, and lactation periods and clinical signs were recorded at the same time and more frequently when warranted. After litter production, all P0 and P1 parental rats and 20 P1 and F1 weanlings/sex/concentration underwent gross postmortem examination.
Litter observations:
P1 and F1 pup body weights and clinical observations were recorded on postpartum days 0, 4, 7, 14, 21 and 25.
Postmortem examinations (parental animals):
Reproductive organs and pituitary gland were collected from each adult animal. Tissues from adult rats in the control and 7000 ppm groups of both generations were examined microscopically.
Postmortem examinations (offspring):
Tissues from adult rats in the control and 7000 ppm groups of both generations were examined microscopically.
Statistics:
In general, sequential trend testing was applied to the data of each parameter. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. Due to limitations of the data collection and reporting system, in the reproductive toxicity study, adult body weight and food consumption data were analyzed by pair-wise comparisons. The level of significance selected for all analyses was p ≤ 0:05.
Parametric analyses were used to compare continuous data such as adult body weight and food consumption data among study groups. Linear contrast of means from One-way Analysis of Variance (ANOVA) was the method of analysis in the developmental toxicity studies; in the
reproductive toxicity study, Dunnett's test followed the ANOVA. Litter-related continuous data were analyzed by a nonparametric method Jonckheere's trend test. For litter parameters, the proportion of affected fetuses per litter or the litter mean was used as the experimental unit for statistical evaluation. Where the data were tied, exact p values were calculated using permutation methodology. Fetal and pup weight data were analyzed by an Analysis of Covariance (covariates: litter size, sex ratio) followed with a linear contrast of the least square means. Discrete data, such as the incidences of clinical observations and reproductive indices, were evaluated by the Cochran-Armitage test for trend. Since tissues or organs were not microscopically evaluated in all groups, the incidences of microscopic observations were analyzed by the Fisher's exact test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The most sensitive indicator of toxicity was the animals' response to a sound stimulus while in the exposure chambers. With a few exceptions, animals exposed to 2000 or 7000 ppm exhibited a transient diminished or absent response to a sound stimulus during each exposure session, beginning exposures 16 and 15, respectively. The diminished or absent alerting responses were interpreted to be a compound-related sedative effect.
Males of both generations exposed to 2000 and 7000 ppm and females of both generations exposed to 7000 ppm cyclohexane, exhibited statistically significantly increased incidences of fur stains and wetness presumably related to salivation. The signs observed in the 2000 and 7000 ppm groups of the reproductive toxicity study were considered to be compound related, but were considered not to be toxicologically important.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: There were no compound-related statistically significant reductions in mean body weight, mean body weight gain.
Females: Mean body weight and overall body weight gain were statistically significantly reduced for 7000 ppm P0 female rats by the end of the premating period (weight gain approximately 87% of control). Mean body weight and weight gain were generally also reduced throughout the study for P1 female rats in the 7000 ppm group , but of a lesser magnitude than that of the P0 females (weight gain approximately 92% of control).
Overall mean body weight gain during gestation was not affected by cyclohexane exposure in the P0 females. The reduction in absolute gestational body weight in P0 females was due to pre-existing body weight deficits established during the premating period.
During the lactation period, overall body weight gains of the P0 females in the 2000 ppm and 7000 ppm exposure groups were greater than that of the P0 control group, which lost weight.
More details presented in Figures 1-4 attached. (See attachments).
Figure explanations:
P1 means first parental generation which corresponds to P0 in the text above.
F1 means second parental generation which corresponds to P1 in the text above.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: There were no compound-related statistically significant reductions in overall mean daily food consumption, or overall mean food efficiency (g body weight gain/g food eaten) for P0 male rats of any concentration the study.
Females: Food consumption was similar between control and treated rats in P0 and P1 females during the premating period.
Gestation period: Food consumption in the 7000 ppm P0 females was less than in control females during gestation days 0-7; no difference was observed for the P1 females.
There were no differences in food consumption during lactation for either the P0 or P1
females, although the P0 7000 ppm females had better food efficiency than their control group, attributable to the difference in body weight gains between the two groups.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):

Females: Food efficiency was reduced in 7000 ppm females in both generations.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no biologically or statistically significant dose-related differences in mating, fertility, or gestation indices, implantation efficiency, or gestation length in either the P0 or P1 generation.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: significant effect at 7000 ppm, thus NOEL determined at 2000 ppm

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males of both generations exposed to 2000 and 7000 ppm and females of both generations exposed to 7000 ppm cyclohexane, exhibited statistically significantly increased incidences of fur stains and wetness presumably related to salivation. The signs observed in the 2000 and 7000 ppm groups of the reproductive toxicity study were considered to be compound related, but were considered not to be toxicologically important.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: Mean body weight was generally statistically significantly reduced throughout the study for P1 male rats in the 7000 ppm group. This was apparently due to preexisting body weight differences established as pups during the lactation period, as overall mean body weight gain.
Females: Mean body weight and weight gain were generally also reduced throughout the study for P1 female rats in the 7000 ppm group , but of a lesser magnitude than that of the P0 females (weight gain approximately 92% of control).
Overall mean body weight gain during gestation was not affected by cyclohexane exposure in the P1 females.
During the lactation period, overall body weight gains of the P0 females in the 2000 ppm and 7000 ppm exposure groups were greater than that of the P0 control group, which lost weight. A similar difference did not occur during the lactation period of P1 females.
See more details in the Figures 1-4 attached.
Figure explanations:
P1 means first parental generation which corresponds to P0 in the text above.
F1 means second parental generation which corresponds to P1 in the text above.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male: Mean daily food consumption and mean daily food efficiency did not differ significantly from controls for P1 male rats exposed to cyclohexane, during the premating period.
Females: Food consumption was similar between control and treated rats in P1 females during the premating period. Food efficiency, however, was reduced in 7000 ppm females in both generations.
Gestation period: Food consumption in the 7000 ppm P0 females was less than in control females during gestation days 0-7; no difference was observed for the P1 females.
There were no differences in food consumption during lactation for P1 females.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Females: Food efficiency, however, was reduced in 7000 ppm females in both generations.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no biologically or statistically significant dose-related differences in mating, fertility, or gestation indices, implantation efficiency, or gestation length in either the P0 or P1 generation.

Effect levels (P1)

Key result
Dose descriptor:
NOEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: significant effect at 7000 ppm, thus NOEL at 2000 ppm.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
With the exception of the mean percent born alive of P1 litters, there were no statistically significant dose-related trends in mean number of implantation sites, mean number of pups/litter, or any survival indices for both P1 and F1 litters throughout lactation. For P1 litters of 7000 ppm
females, mean percent born alive was statistically significantly reduced.
However, that reduction was not repeated in F1 litters, and was considered to be spurious; the value (98.1%) fell above the mean of our historical control data (mean 97.5%; range of 92.5% - 100%).
See Table IV - VI attached.
P1 means first parental generation which corresponds to P0 in the text above.
F1 means second parental generation which corresponds to P1 in the text above.
F2 means litter coming from P1 which corresponds to F1 in the text above.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for P1 litters and F1 litters of the 7000 ppm group. There were no statistically significant dose-related differences in the incidences of any clinical signs among P1 or F1 litters.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
7 000 ppm
Treatment related:
yes

Any other information on results incl. tables

Еhe overall mean analytically-determined chamber concentrations were as targeted: 500, 2000, and 7000 ppm cyclohexane (2 significant figures). Analyses of cyclohexane performed before, during, and after the conduct of the studies indicated that the cyclohexane was stable over the duration of the 3 studies and was 99.99% pure. Mean values for temperature, relative humidity, oxygen, and air flow were similar among the 4 chambers. Mean daily chamber temperatures ranged from 18-28oC. Mean daily humidity ranged from 29-71%. Mean daily chamber air flow ranged from 210-360 L/min. Mean daily oxygen concentration ranged from 19-21%. The environmental conditions in the exposure chambers were considered to be within acceptable ranges for the health and comfort of the animals and were considered to be adequate to meet the study requirements.

Applicant's summary and conclusion

Conclusions:
The systemic toxicity no-observed-effect level (NOEL) was 500 ppm and the reproductive NOEL was 2000 ppm. The reproductive NOEL was based solely on the decreased pup weights in both the P1 and F1 generations observed at 7000 ppm.
Executive summary:

In the two-generation reproduction study, parental effects included statistically significantly lower mean body weight, overall mean body weight gain, and overall mean food eficiency for P0 and P1 females of the 7000 ppm level and statistically significantly lower mean body weight for P1 males of that level. Adult rats exposed to 2000 ppm cyclohexane and above exhibited a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Mean pup weight was statistically significantly lower than control from lactation day 7 throughout the remainder of the 25-day lactation period for both P1 and F1 7000 ppm litters. Changes observed at 500 ppm were either considered not to be compound related or not adverse. Therefore, the systemic toxicity no-observed-effect level (NOEL) was 500 ppm and the reproductive NOEL was 2000 ppm. The reproductive NOEL was based solely on the decreased pup weights in both the P1 and F1 generations observed at 7000 ppm. In the developmental toxicity studies, only the rats showed evidence of maternal toxicity. For rats in the 7000 ppm group, statistically signiffcant reductions were observed in overall maternal body weight gain and overall maternal food consumption for the treatment period. Rats exposed to 2000 ppm cyclohexane and above again exhibited
a transient diminished or absent response to a sound stimulus while in the chambers during exposure. Therefore, for rats, the maternal no-observed-effect level (NOEL) was 500 ppm.