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Toxicological information

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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
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
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
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
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
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.
Dose descriptor:
NOAEC
Effect level:
>= 500 - <= 2 000 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
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
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.
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: 24,080 mg/m3
Reproductive effects observed:
not specified

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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
24 080 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate information is available to characterise the effects of cyclohexane on fertility following inhalation exposure.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A two generation study was conducted to assess the reproductive and developmental toxicity of cyclohexane in the Crl: CD BR rat. Groups of rats were exposed to nominal concentrations of 0, 500, 2000 or 7000 ppm cyclohexane (whole body).  Effects in parents included statistically significantly lower mean body weight, lower overall mean body weight gain, and lower overall mean food efficiency for P1 and F1 females exposed to 7000 ppm. In F1 males only mean body weights were affected.  Adult rats exposed to 2000 or 7000 ppm cyclohexane were observed to have a transient diminished or absent response to a sound stimulus (in chambers during exposure). In the 7000 ppm group mean pup weight was statistically significantly lower than control from lactation day 7 to the end of the lactation period on day 25 or both F1 and F2 litters. It was concluded that changes observed at 500 ppm were either not compound related or not adverse. Therefore, it was concluded that the systemic-toxicity no-observed-effect concentration (NOAEC) was 500 ppm.  The reproductive NOAEC was 2000 ppm on the basis of decreased pup weights in both the F1 and F2 generations) at 7000 ppm. As pup bodyweights in all groups were similar to control at birth the effect of treatment is judged to be systemic, not reproductive. It is concluded that the NOAEC for effects on male and female fertility was 7000 ppm (24,080 mg/m3), the highest dose tested.


Short description of key information:
In a 2-generation study, with a top dose of 7000 ppm (24,080 mg/m3), no effect was seen on reproductive parameters.

Justification for selection of Effect on fertility via inhalation route:
No effect on reproductive parameters in a 2-generation study at 7000 ppm (24,080 mg/m3, the highest dose tested)

Effects on developmental toxicity

Description of key information
In the 2-generation study, no effect was seen on reproductive parameters. A slight decrease of the pups body weight was observed at 7,000 ppm, this decrease being accompanied by slight maternal toxicity. A NOAEC of 2,000 ppm (6,880 mg/m3) can be determined for pups whereas a NOAEC of 500 ppm (1,720 mg/m3) can be derived for maternal toxicity. 
No toxic effect was observed in the foetuses in two developmental studies performed in rats and in rabbits. Toxic effects were noted in the dams and were consistent with those observed in the other studies (narcotic effects). The highest dose tested in these studies (7,000 ppm (24,080 mg/m3))
can be considered to be the NOAEC for foetuses with a NOAEC of 500 ppm (1,720 mg/m3) for dams.
Link to relevant study records
Reference
Endpoint:
developmental 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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- Temperature: 23±2°C
- Humidity: 50±10%
- Air changes: Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: not reported
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
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.

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)
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).
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:
CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after exposure, otherwise once/day

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

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- 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.
Statistics:
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.
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
Dose descriptor:
NOAEC
Effect level:
500 - 2 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
7 000 ppm
Basis for effect level:
other: developmental toxicity
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.
Dose descriptor:
NOAEC
Effect level:
7 000 ppm
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Mean maternal bodyweight gain (g) selected timepoints

Treatment

Days 0 to 6

Days 6 to 16

Days 6 to 21 (b)

0 ppm

18.7

64.2

49.6

0# ppm

22.4

32.1

12.5

500 ppm

22.3

60.1

43.0*

2000 ppm

24.6

57.2*

43.0*

7000 ppm (b)

22.1

44.2*

37.1*

 # 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

Conclusions:
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).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
24 080 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate information is available to characterise the effects of cyclohexane on foetal development following inhalation exposure.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
The developmental toxicity of cyclohexane was assessed in Crl: CD BR rats. The animals were exposed to 0, 500, 2000, or 7000 ppm cyclohexane vapour (whole body). Statistically significant reductions in overall and adjusted maternal body weight gain were observed in dams exposed to 7000 ppm cyclohexane 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 500 ppm (based upon transient sedation) or 2000 ppm (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. The developmental toxicity of cyclohexane was also assessed in a study with Hra: (NZW)SPFrabbits. The animals were exposed to 0, 500, 2000 or 7000 ppm cyclohexane (whole body). No compound-related maternal effects were observed at concentration levels of 7000 ppm and below. The maternal NOAEC for rabbits was 7000 ppm. No compound-related evidence of developmental toxicity was observed at any test concentration. It was concluded that the developmental NOAEC for rabbits was 7000 ppm (24,080 mg/m3), the highest concentration tested and the highest concentration permissible under national fire protection association standards.

Justification for selection of Effect on developmental toxicity: via inhalation route:
No effect on foetal development following exposure of pregnant rats and rabbits to 7000 ppm cyclohexane vapour (24,080 mg/m3, the highest dose tested)

Toxicity to reproduction: other studies

Additional information
No other reproduction studies are available

Justification for classification or non-classification

Based on the results of the 2 -generation and developmental toxicity study results and according to EU criteria, cyclohexane does not warrant classification for reproductive or developmental toxicity according to CLP.

Additional information