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Developmental toxicity / teratogenicity

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

Data source

Reference Type:

Materials and methods

Test guidelineopen allclose all
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
not specified
equivalent or similar to guideline
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
not specified
GLP compliance:
Limit test:

Test material

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

other: Crl:CD BR
Details on test animals or test system and environmental conditions:
- Age at study initiation: Approximately 11 weeks
- Housing: Animals were housed individually in suspended, wire-mesh, stainless steel cages.
- Diet: Purina "Certified Rodent Checkers" was available ad libitum, except during exposures to all rats other than those in the pair-fed control group of the developmental toxicity study. Beginning with exposure, each animal in the pair-fed control group received an amount of Purina Certified Rodent Checkers approximately equal to the cumulative average amount of food consumed by the high-concentration group (7000 ppm) animals on the corresponding gestation day
- Water: tap water ad libitum except during exposures

- Temperature: 23±2°C
- Humidity: 50±10%
- Air changes: 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
other: air
Details on exposure:
- 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.

- 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)
Analytical verification of doses or concentrations:
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).
Details on mating procedure:
- Impregnation procedure: co-habitation
- If cohoused:
- M/F ratio per cage: 1:1

- Proof of pregnancy: no details. The day that copulation was confirmed was designated as day 0 of pregnancy
Duration of treatment / exposure:
Assumed-pregnant rats (25/concentration level) were exposed on days 6-15 of gestation (6-15G).
Frequency of treatment:
6 hours/day
Duration of test:
exposed for 10 days on days 6-15 of gestation, killed on day 21 of gestation
No. of animals per sex per dose:
25 per concentration level
Control animals:
yes, sham-exposed
Details on study design:
A pair-fed control group was set up for the 7000 ppm treatment group. Beginning with exposure, each animal in the pair-fed control group received an amount of Purina Certified Rodent Checkers approximately equal to the cumulative average amount of food consumed by the high-concentration group (7000 ppm) animals on the corresponding gestation day. Each rat received approximately 150 grams per day of Purina Certified Rat Diet HF #5325; feed was not available during exposure.


Maternal examinations:
- Time schedule: before and after exposure, otherwise once/day

- Time schedule for examinations: once per day during exposure, weekly at all other times



- Sacrifice on gestation day 21 (maternal animals killed, organs of the thoracic and abdominal cavities examined grossly. The method of euthanasia was carbon dioxide asphyxiation).
Ovaries and uterine content:
The uterus of each animal was removed and opened. The types of implants (live and dead foetuses, and resorptions) were counted and their relative positions were recorded.
Fetal examinations:
The foetuses were euthanatized by decapitation or by intraperitoneal injection of sodium pentobarbital. They were weighed, sexed, and examined for external, visceral and skeletal alterations.
For all analyses the level of significance was p < 0.05. The data for each parameter were subject to sequential trend testing. Continuous data were compared using parametric analyses. The method of analysis was linear contrast of means from One-way Analysis of Variance (ANOVA). The nonparametric method, Jonckheere's trend test was used to analyse litter-related continuous data. The proportion of affected foetuses per litter or 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 foetal weights. The Cochran-Armitage test for trend was used to evaluate discrete data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no unscheduled deaths. Rats exposed to 2000 or 7000ppm exhibited a transient diminished or absent alerting response during each exposure session; this effect was not seen in rats exposed to 500ppm. Overall mean body weight gain for the exposure period (days 6-16) was statistically significantly reduced for rats exposed to 7000 ppm (69% of control) and mean daily food consumption was 89% of control. Mean body weight gain for the exposure and post-exposure period (Days 6-21) calculated using the final body weight minus the gravid uterine weight) was also statistically significantly reduced (75% of control). There was judged to be no effect of exposure to 2000 or 500ppm on maternal bodyweight.
A similar reduction in weight gain was seen in the pair-fed animals. There were no post-mortem findings that were considered indicative of a treatment-related effect

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
500 - 2 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
7 000 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related differences in pregnancy rate, early delivery rate, abortion rate, total resorption rate, mean number of implantations per litter, the mean number of live foetuses per litter or sex ratio. There were no dead foetuses, nor was there any difference in the incidence of early, late or total resorptions. Foetal weight was unaffected by treatment. There were no effects on the incidence of foetal malformations or variations.

Effect levels (fetuses)

Dose descriptor:
Effect level:
7 000 ppm
Basis for effect level:
other: teratogenicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mean maternal bodyweight gain (g) selected timepoints


Days 0 to 6

Days 6 to 16

Days 6 to 21 (b)

0 ppm




0# ppm




500 ppm




2000 ppm




7000 ppm (b)




 # pair fed control b- ANOVA and Dunnett’s test

 significant difference from control (ANOVA and Dunnett’s test); p±0.05

* significant trend (linear contrast of means from ANOVA); p±0.05

b bwt gain on GD6-21 after correction for gravid uterus weight

Applicant's summary and conclusion

Cyclohexane was not a developmental toxin in female rats exposed during pregnancy. The foetal NOAEC was 7000 ppm, and the maternal NOAEC was 500 ppm (based upon transient sedation) or 2000 ppm (based upon significant reductions in absolute and adjusted body weight gain).
Executive summary:

The developmental toxicity of cyclohexane was assessed in Crl:CD BR rats. The animals were exposed whole-body to nominal atmospheric concentrations of 0, 500, 2000, or 7000 ppm cyclohexane vapour. For rats in the 7000 ppm group, statistically significant reductions were observed in overall and adjusted maternal body weight gain while a transient diminished or absent response to a sound stimulus was apparent at 2000 ppm. Therefore the maternal no-observed-adverse-effect concentration (NOAEC) was 500ppm (1,720 mg/m3) (based upon transient sedation) or 2000 ppm (6,880 mg/m3) (based upon significant reductions in overall and adjusted body weight gain).  No compound-related evidence of developmental toxicity was observed at any test concentration, equivalent to a NOAEC of 7000 ppm (24,080 mg/m3).