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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study conducted according to a previous guideline version. Therefore, comparable to guideline study with acceptable restrictions. Only 10-day treatment period (Days 7-16 of gestation). Refer to endpoint summary Toxicity to reproduction and IUCLID Section 13 for reporting and justification of the analogue approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 21 May 1981
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only 10-day treatment period (Days 7-16 of gestation)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyclohexane
- Molecular formula: C6H12
- Molecular weight: 84.16
- Smiles notation: C1CCCCC1
- InChl: 1S/C6H12/c1-2-4-6-5-3-1/h1-6H2
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: > 99.9% by analysis

Test animals

Species:
rat
Strain:
other: Crl:CD BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc. Raleigh, USA
- Age at study initiation: females: approx. 63 days; males: approx. 77 days
- Weight at study initiation: females: 175.0 - 224.1 g; males: 310.2 - 369.1 g
- Housing: individually in suspended, wire-mesh, stainless steel cages
- Diet: Purina Certified Rodent Checkers (approx. equal to the average amount of feed consumed by the high-concentration group on the corresponding gestation day)
- Water: ad libitum, except exposure period
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers (NYU style) were constructed of stainless steel and glass with a nominal volume of 1.4 m³.
- System of generating vapours: Atmospheres were generated by metering the liquid test substance into a heated glass Instantherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the test substance vapour into the inhalation chamber air supply. The chamber concentration of the test substance 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 approx. the same flowrates as those used in the exposure chambers. Chamber atmospheres were exhausted into the main plenum exhaust system and emitted into the atmosphere as allowed by permit.
The chamber distribution of test substance vapour was determined prior to animal exposures in the low- and high-concentration exposure chambers. 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) for inhalation toxicology testing. A tangential feed at the chamber inlet promoted gas mixing and uniform chamber distribution of vapour.
- Mean airflow: 320 - 330 L/min
- Mean temperature in exposure chamber: 21 - 22 °C
- Mean relative humidity: 48 - 52%
- Oxygen concentration: 20 - 21%

TEST ATMOSPHERE
- Brief description of analytical method used: The atmospheric concentration was determined by gas chromatography at approx. 15-min intervals during each 6-hour exposure. Gas samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed.
The nominal atmospheric concentration was determined daily for each chamber by taking the total amount of test substance delivered to the inhalation equipment divided by the total volume of air.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmosphere samples were directly injected into a gas chromatograph equipped with a flame ionization detector for analysis of test substance concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: each female was individually housed with a male
- M/F ratio per cage: 1/1
- Length of cohabitation: 3 days
- Proof of pregnancy: vaginal plug; referred to as day 1 of pregnancy (day 1G)
Duration of treatment / exposure:
Day 7 - 16 of gestation (d7 - 16G)
Frequency of treatment:
6 h/day, 7 days/week
Duration of test:
The dams were sacrificed on gestation day 22.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 2000 and 7000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
25 mated females
Control animals:
yes, sham-exposed
other: pair-fed control
Details on study design:
- Dose selection rationale:
The high concentration level was selected on the basis of a pilot developmental toxicity study conducted at levels of 0, 3000, 6000 and 9000 ppm and knowledge of the physical/chemical properties of the test substance. The low concentration was selected because this value exceeds the threshold limit value for human exposure (300 ppm) and was expected to cause no toxicological effects. The intermediate concentration level (2000 ppm) was selected to provide approximately equal spacing between the high and low concentations on a log scale.
- Other:
Because of the reduction in food consumption observed in the pilot study, a pair-fed control group was included as part of the study design.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During each exposure, rats were observed for mortality approx. every hour, and were assessed for their response to an altering stimulus.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before beginning of the study, daily on days 1-6G and 17-22G (of gestation) and twice per day on days 7 – 16G.

BODY WEIGHT: Yes
- Time schedule for examinations: at the day of arrival, twice during the first week, weekly before beginning of the study, day 1, 7-17 and 22 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, individual feeder weights were measured daily. Beginning with exposure, each animal in group II (pair-fed control) received an amount of food approx. equal to the cumulative average amount of food consumed by group V (high concentration) animals.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22
- Organs examined: viscera, weight of intact and empty uterus of each dam having at least one viable foetus, corpora lutea (count for each ovary)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- Number, Location, Sex, and Condition: Yes: all per litter
- Foetal weight: Yes: all per litter
- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: For each litter, the first live foetus and every other live foetus thereafter was examined for visceral alterations. Those foetuses were decapitated before, proceeding with visceral examinations. In addition, all live foetuses with malformations visible at external examination and all stunted foetuses were examined for soft tissue alterations, and the sex verified. Retarded renal development was classified using the schema of Woo an Hoar.
- Head examinations: Yes: After fixation in Bouin´s, the heads of decapitated foetuses were examined, based on the method of Barrow and Taylor.
- Skeletal examinations: Yes: After fixation, the alizarin-stained skeletons were examined and skeletal alterations were recordced for all live foetuses, excluding the foetal heads fixed in Bouin´s.
Statistics:
Sequential trend testing (excluding the pair-fed control group) was applied to the data for each parameter as tabulated below. If a significant dose-response was detected, data from the top dose group was exculuded and the test repeated until no significant trend was detected. For litter parameters, the proportion of affected foetuses per litter or the litter mean was used as the experimental unit for statistical evaluation. The level of significance selected was p <= 0.05. Where the data were tied and the standard large sample version of Jonckheere´s test was not applicable, exact p-values were calcualted using permutation methodology.
- Linear contrast of means from ANOVA: maternal weight, maternal weight changes and maternal food consumption
- Jonckheere´s test: live foetuses, dead foetuses, resorptions, nidations, corpora lutea and incidence of foetal alterations
- Cochran-Armitage test: incidence of pregnancy, clinical observations, maternal mortality, females with total resorptions and early deliveries
- Linear contrast of least square means from ANCOVA: foetal weight (covariates: litter size, sex ratio) and sex ratio (covariate: litter size)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
No maternal mortality occurred during the study.

CLINICAL SIGNS
- 7000 ppm: stain chin, salivation (lasting only 10-15 min after the animals were removed from the chambers)
These findings were considered compound-related but not toxicologically important.
- 2000 and 7000 ppm: compound-related effects on the alerting response determined during exposure were observed. Rats of these groups exhibited a diminished response or no resonse during each exposure session. This was interpreted to be a transient, but toxicologically important compound-related sedative effect.

BODY WEIGHT
- 7000 ppm: Overall mean body weight gain for the exposure period (day 7-17G) was statistically significantly reduced (approx. 69% of control). Mean body weight gain for the exposure and postexposure period (day 7-22G) calculated using the adjusted final body weight, was also statistically significantly decreased (approx. 75% of control). By the end of the exposure period (day 17G) absolute maternal body weight had become statistically signifcantly reduced (approx. 95% of control).
- 2000 ppm: Mean maternal body weight gain was statistically significantly reduced for the exposure period (day 7-17G) (approx. 89% of control). However, that reduction was considered not to be adverse since the value (57.2 g) fell above the mean value for control data from previous studies conducted at the same laboratory over several years.
- 500 and 2000 ppm: Mean adjusted body weight gain for the exposure and posteexposure period (day 7-22G) was statistically significantly decreased (approx. 87% of control). However, those reductions were considered not to be adverse since the value (43 g) fell above the mean value for control data from previous studies conducted at the same laboratory over several years.

FOOD CONSUMPTION
- 7000 ppm: Changes observed in food consumption corresponded with changes observed in body weight gain. The overall mean daily food consummption during the exposure preiod (day 7-17G) was statistically significant lower than control rats (approx. 89% of control)

POSTMORTEM FINDINGS
There were no postmortem findings recorded for adult female rats suggestive of compound-related effects.

REPRODUCTIVE PARAMETERS
There were no statistically significant differences between control and treatment groups in the pregnancy rate, early delivery rate, total resoprtion rate, the mean number of live fetuses per litter, or sex ratio.
A slight but statistically significant decrease in the number of implantations was also observed. Although the decreased mean was below the range of the historical control data of the test facility, the mean number of corpora lutea was comparable to that of the control group, thus suggesting pre-implantation loss. Since there was no exposure during pre-implantation, this finding was considered not to be substance-related.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
1 720 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
7 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEC
Effect level:
24 080 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
MORTALITY
There were no dead foetuses and no statistically significant differences between control and treatment groups in early, late,or total resoprtions.

BODY WEIGHT
There were no statistically significant difference between control and treament groups in mean foetal weight.

MALFORMATIONS / VARIATIONS
No compund-related effect on the incidence of foetal malformations was observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Assumed-pregnant rats (25/concentration) were exposed whole-body to cyclohexane at 500, 2000 and 7000 ppm (corresponding to 1720, 6880 and 24080 mg/m³). Sham-exposed control groups were included. Rats were exposed for 6 h/day on Days 7-16 of gestation. On Day 22 of gestation, animals were sacrificed for gross pathological examination. Uteri were removed and opened. The types of implants (live and dead fetuses and resorptions) were counted and their relative position recorded. Live fetuses were weighed, sexed and examined for external, visceral and skeletal alterations.
Substance-related adverse effects observed at 2000 and 7000 ppm. At both concentrations, a generally diminished or absent response to a sound stimulus was noted during the 6 h exposure period. A statistically significant reduction in overall maternal body weight gain and overall maternal food consumption during the treatment period was seen at 7000 ppm. At this concentration, a slight but statistically significant decrease in the number of implantations was also observed. Although the decreased mean was below the range of the historical control data of the test facility, the mean number of corpora lutea was comparable to that of the control group, thus suggesting pre-implantation loss. Since there was no exposure during pre-implantation, this finding was considered not to be substance-related.
There were no substance-related differences between control and treated groups in fertility, number of resorptions, number of live fetuses, sex ratio and mean fetal weight. No substance-related effects were observed on the incidence of fetal malformations and variations.
Based on the effects observed at 2000 and 7000 ppm, the maternal NOAEC was 500 ppm , equivalent to 1720 mg/m³. No evidence of developmental toxicity was observed at any concentration. Therefore, the developmental NOAEC was 7000 ppm (24080 mg/m³).
Cyclohexane had no effects on intrauterine development.