Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexane
EC Number:
203-806-2
EC Name:
Cyclohexane
Cas Number:
110-82-7
Molecular formula:
C6H12
IUPAC Name:
cyclohexane
Details on test material:
Name of test material (as cited in study report): cyclohexane
- Supplier: Phillips Petroleum Company, Sweeney Refinery, Sweeney, Texas, USA
- Physical state: liquid
- Analytical purity: >99.9%

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Housing: Animals were housed individually in suspended, wire-mesh, stainless steel cages. 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.
- Diet: Purina "Certified Rodent Checkers" was available ad libitum, except during exposures
- Water: tap water ad libitum, except during exposures

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2°C
- Humidity: 50±10%
- Air changes (per hr): not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass exposure chambers with a nominal internal volume of 1.4 m3 (total body volume of the test animals did not exceed 5% of the chamber volume). A tangential feed at the chamber inlet promoted gas mixing and uniform chamber distribution of vapour.
- Cyclohexane atmosphere generation: the liquid cyclohexane was metered into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapour into the inhalation chamber air supply. The chamber concentration was controlled by varying the amount of the metered liquid evaporated in the chamber air stream. Nitrogen and air were passed through the control chamber at approximately the same flow rates as those used in the exposure chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography equipped with a flame ionization (at approximately 15-minute intervals during each 6-hour exposure)
- Samples taken from breathing zone: yes (atmosphere samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: not reported
- Proof of pregnancy: not reported. The day copulation was confirmed was designated day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The atmospheric concentration of cyclohexane was determined by gas chromatography at approximately 15 minute intervals during each 6-hour exposure. The results of these determinations indicated the distribution of cyclohexane vapour was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position).
Duration of treatment / exposure:
The P1 generation was exposed for at least 10 weeks prior to mating within their respective treatment groups, and allowed to deliver and rear their offspring until weaning (postpartum day 25). Pregnant females were exposed daily during days 0-20 of gestation. They were not exposed from day 21 of gestation until day 4 of lactation. Females resumed their daily exposure regimen from day 5 of lactation until weaning of litters. They were returned to their litters each day after exposure. Males continued to be exposed 5 days/week until sacrifice. Neonates were not exposed to cyclohexane by inhalation during lactation.
At least 11 weeks after weaning, thirty selected F1 rats/sex/ group were bred within their respective treatment groups to produce F2 litters, following the same regime.
Frequency of treatment:
6 hours/day, 5 days/week (excluding holidays)
Details on study schedule:
At least 11 weeks after weaning 30 F1 rats/sex/group were bred within their respective treatment groups to produce F2 litters
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 (air), 500, 2000, 7000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0 (air), 500, 2000, 7000 ppm
Basis:
other: overall mean measured
No. of animals per sex per dose:
30 /sex/concentration
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Prior to the initiation of each exposure, during exposure, and during the time required to clear the chambers of test substance, the groups of animals within exposure levels were observed for a normal, diminished, or hyper-responsive alerting behaviour in response to a standardized auditory stimulus. All adult P1 and F1 rats were weighed weekly during the premating, gestation, and lactation periods and clinical signs were recorded at the same time.
Litter observations:
F1 and F2 pup body weights and clinical observations were recorded on postpartum days 0, 4, 7, 14, 21 and 25.
Postmortem examinations (parental animals):
At day 25 post partum all P1 and F1 parental rats underwent gross postmortem examination. The method of euthanasia was carbon dioxide asphyxiation and exsanguination. Reproductive organs and pituitary gland were collected from each adult animal. Tissues from P1 and F1 adult rats in the control and 7000 ppm groups of both generations were examined microscopically.
Postmortem examinations (offspring):
20 F1 and F2 weanlings/sex/concentration underwent gross postmortem examination. The method of euthanasia was carbon dioxide asphyxiation and exsanguination.
Statistics:
For all analyses the level of significance was p < 0.05. The data for each parameter were subject to sequential trend testing. Pair-wise comparisons were used to analyse adult body weight and food consumption data. Continuous data were compared using parametric analyses. The method of analysis was linear contrast of means from One-way Analysis of Variance (ANOVA) followed by Dunnett's test. The nonparametric method, Jonckheere's trend test was used to analyse litter-related continuous data. The litter mean was used as the experimental unit for statistical evaluation of litter parameters. Exact p values were calculated using permutation methodology where the data were tied. Analysis of Covariance (covariates: litter size, sex ratio) followed by a linear contrast of the least square means was used to analyse pup weights. The Cochran-Armitage test for trend was used to evaluate discrete data. Fisher's exact test was used to analyse microscopic observations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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 at 2000 and 7000 ppm and females at 7000 ppm had increased incidences of fur stains and wetness. These signs were considered to be compound related, but not to be toxicologically important.
A statistically significant reduction in mean body weight and overall mean body weight gain was present in the P1 and F1 females exposed to 7000 ppm. i.e. at the end of the premating period weight gain was 87% of control for P1 females and 92% of controls for F1 females. There was no effect of 7000 ppm on the bodyweight of males but mean bodyweight was generally statistically significantly reduced throughout the study in the F1 males. Food efficiency was reduced in 7000 ppm females in both generations.
There were no biologically or statistically significant dose-related differences in mating, fertility or gestation indices, implantation efficiency or gestation length in either the P1 or F1 generations.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
>= 500 - <= 2 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

There was no significant effect on mean number of implantation sites, mean number of pups/litter or survival indices for the F1 and F2 litters. Mean pup weight was statistically significantly reduced from post-partum day 7 through-out the remainder of the 25 day lactation period for F1 and F2 litters from the 7000 ppm group.

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEC
Generation:
F2
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mean pup weights (g) selected timepoints

 

Day 0

Day 7

Day 14

Day 21

Day 25

F1 Generation

 

 

 

 

 

0 ppm

6.7

16.2

30.0

48.5

67.5

500 ppm

6.7

16.2

29.9

48.5

67.8

2000 ppm

6.7

16.3

29.7

48.3*

68.3

7000 ppm

6.6

15.1*

26.5*

43.1*

62.2*

F2 Generation

 

 

 

 

 

0 ppm

6.4

16.3

31.0

50.0

69.3

500 ppm

6.6

16.0

30.2

48.3

67.1

2000 ppm

6.3

15.3

28.9

46.4

65.6

7000 ppm

6.3

14.3*

26.2*

42.8*

61.3*

* Statistically significant difference from control (p 0.05) by Analysis of Covariance with litter size and sex ratio as covariates

Applicant's summary and conclusion

Conclusions:
Parental effects were restricted to transient sedation from study day 15-16 (NOAEC 500 ppm) and body weight effects (NOAEC 2000 ppm). There was, however, no adverse effect on reproductive function in male or female rats following exposures up to 7000 ppm.
Executive summary:

A two-generation reproduction study was conducted to assess the reproductive and developmental toxicity of cyclohexane. Rats of the Crl:CD BR strain were exposed whole-body to atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane. Statistically significantly lower mean body weight, overall mean body weight gain, and overall mean food efficiency were recorded for the parent P1 and F1 females exposed to 7000 ppm although a statistically significantly lower mean body weight was recorded for parent F1 males only. During exposure, the adult rats exposed to 2000 ppm cyclohexane or more exhibited a transient diminished or absent response to a sound stimulus.

 

There was a statistically significant lower mean pup weight, from day 7 through to day 25 of lactation, for F1 and F2 litters exposed to 7000 ppm. The no-observed-effect concentration (NOAEC) for systemic toxicity was 500 ppm (1,720 mg/m3) and the NOAEC for reproductive toxicity (based on the decreased pup weights in the F1 and F2 generations) was 2000 ppm. The NOAEC for effects on male and female fertility was 7000 ppm (24,080 mg/m3), the highest concentration tested.