2,2,4-trimethylpentane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no data available for 2,2,4-trimethylpentane. However, data in rats is available for structural analogues, hexane and cyclohexane and presented in the dossier. This data is read across to 2,2,4-trimethylpentane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Cyclohexane

Two-Generation Reproduction Toxicity Study (OECD TG 416)

NOAEC (Rat) >= 7000 ppm (24080 mg/m³)

Commercial Hexane

Two-Generation Reproduction Toxicity Study (OECD TG 416)

NOAEC (rat) = 9000 ppm (31680 mg/m³)

Additionally, in order to comply with standard information requirements for Annex X substances, an OECD 443 test is proposed for structural analogue, Hydrocarbon, C7-C9, isoalkanes (EC# 921-728-3). This read across is based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in Section 13.2 of the dossier.This endpoint will be updated subsequent to ECHA's approval of the testing proposal and availability of data upon completion of the study.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restriction because this study was performed in accordance with GLP and appeared to closely followed OECD 416.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P males) 8 weeks; (P females) 8 weeks
- Housing: Individually housed except during cohabitation periods in wire mesh cages. Assumed-pregnant females and those without evidence of copulation were individually housed in polycarbonate pans with bedding. During lactation, adult females were housed with their litters in polycarbonate pans with bedding
- Diet (e.g. ad libitum): Ad libitum, Purina Certified Rodent Checkers
- Water (e.g. ad libitum): Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2°C
- Humidity (%): 50 +/- 10%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass and had a nominal internal volume of 1.4 m3. The chamber volume was chosen so that the 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 vapor
- Atmospheres of cyclohexane were generated by metering the liquid test substance into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapor into the inhalation chamber air supply. The chamber concentration of cyclohexane 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-The atmospheric concentration of cyclohexane was determined by gas chromatography at approximately 15-minute intervals during each 6-hour exposure. Chamber-atmosphere samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed and were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization. All samples were chromatographed isothermally at 70°C on an HP-20M Carbowax column. The chamber distribution of cyclohexane vapor was determined prior to animal exposures in the high-concentration exposure chamber and while the study was underway with animals in the low- and high-concentration chambers. The results of these determinations indicated the distribution of cyclohexane vapor was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position).

Details on mating procedure:

No data reported.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentrations were determined by gas chromatography at approximately 15-minute intervals during each 6-hour exposure. Chamber distribution of cyclohexane was determined prior to animal exposures in the high-concentration exposure chamber. Results showed that the distribution of cyclohexane vapour was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position).

Duration of treatment / exposure:

Males and females were exposed prior to mating (at least 10 weeks for the P generation and 11 weeks for the F1 generation). Pregnant females were exposed daily during gestation days 0 through 20; exposure ceased from gestation day 21 until lactation day 4. Exposure resumed on lactation day 5 until litters were weaned. Males continued to be exposed 5 days/week until sacrificed. Neonates were not exposed during lactation.

Frequency of treatment:

6 hours/day, 5 days/week, including holidays

Details on study schedule:

P animals were mated after approximately 10 weeks after exposure. Dams were allowed to deliver and rear pups until weaning (postpartum day 25). After at least 11 weeks after weaning, F1 animals were bread to produce F2 litters. Pups were culled on lactation day 4.

Remarks:

Doses / Concentrations:
0 (air), 500, 2000, and 7000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

P generation: 30 animals/sex/dose
F1 generation: 30 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The high concentration was selected to be 60% of the Lower Explosive Limit; the low concentration was selected because it exceeds the threshold limit for human exposure and was not expected to cause toxicological effects. The mid dose was selected to provide approximately equal spacing between and high and low doses on a log scale.

Positive control:

No positive control was used.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes; 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 behavior in response to a standardized auditory stimulus.

BODY WEIGHT: Yes
- Time schedule for examinations: P and F1 rats were weighed weekly during the premating, gestation, and lactation periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

No data reported.

Sperm parameters (parental animals):

No data reported.

Litter observations:

F1 and F2 pup weights and clinical observations were recorded on postpartum days 0, 4, 7, 14, 21, and 25.

Postmortem examinations (parental animals):

After litter production, all P and F1 parental animals were euthanized by carbon dioxide and exsanguinated. Reproductive organs and pituitary gland were collected from each animal. Tissues from the high-dose and control animals for both generations were examined microscopically.

Postmortem examinations (offspring):

Pups were euthanized by carbon dioxide and exsanguinated.

Statistics:

In general, sequential trend testing was applied to the data of each parameter. If a result was significant, data from the high-dose group was excluded; the test was repeated in until no significant trend was detected. 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.

Among study groups, parametric analyses were used to compare continuous data. Linear contrast was performed by conducting a Dunnett's test followed by ANOVA. Litter-related continuous data were analyzed by Jonckheere's test. Foetal and pup weights were analyzed by an Analysis of Covariance followed with a linear contrast of the least square means. Discrete data were evaluated by the Cochran-Armitage test for trend. The incidence of microscopic observations were analyzed by the Fisher's exact test.

Reproductive indices:

Mating index, fertility index, and mean gestation length were calculated.

Offspring viability indices:

The sex ratio, implantation efficiency, gestation index, day 0-4 viability, lactation index, and litter survival were calculated.

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:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 2000 and 700 ppm, animals exhibited treatment-related transient diminished or absent response to sound stimulus during each exposure session, being at exposures 16 and 15, respectively. Males (P and F1) treated with 2000 and 7000 ppm and 7000-ppm females from both generations were showed significantly increased incidence fur staining and wetness presumably related to salivation. These clinical signs were not considered treatment-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
P males showed no treatment-related body weight changes in any dose group. High-dose F1 males were generally statistically significantly reduced throughout the study. This decrease was considered by the result of pre-existing body weight differences established as pups during the lactation period.

High-dose P females showed a significant decrease in mean body weight and body weight gain by the end of the premating period. High-dose females showed lower body weight and body weight gain, but to a lesser magnitude. Food consumption was similar between control and treated rats for both generations; however, food efficiency was reduced for both generations at the high dose.

During gestation, mean body weight and body weight gain was not affected by treatment. Food consumption for 7000-ppm P females was less than control females during gestation days 0 through 7; no differences were observed for F1 females. During lactation, 2000- and 7000-ppm P females were greater than the control group; these differences were not observed in the F1 generation. There were no differences in food consumption during lactation for either generation; however, 7000-ppm females had better food efficiency than the control group, which was attributable to the difference in body weight gains between the two groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no statistically significant treatment-related differences in mating, fertility, or gestation indices, implantation efficiency or gestation length in either generations.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effects with regard to gross observations in rats of any generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effects with regard to microscopic findings in rats of any generation. An increased incidence of prostatic inflammation in high-dose males in both generations was observed. The severity of this lesion was only in four P and F1 control animals and four high-dose males, and was considered incidental due to its lack of severity and common occurrence in this species.

Key result

Dose descriptor:

other: Parental NOAEC

Effect level:

500 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 1720 mg/m3

Remarks on result:

other: Generation: P and F1

Key result

Dose descriptor:

other: Parental LOAEC

Effect level:

2 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 6880 mg/m3; based on transient treatment-related sedative effect on the rats' alerting response to sound stimulus

Remarks on result:

other: Generation: P and F1

Key result

Dose descriptor:

other: Offspring NOAEC

Effect level:

2 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 6880 mg/m3

Remarks on result:

other: Generation: F1 and F2

Key result

Dose descriptor:

other: Offspring LOAEC

Effect level:

7 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 24,080 mg/m3; decreased mean pup weight from lactation days 7 through 25

Remarks on result:

other: Generation: F1 and F2

Key result

Dose descriptor:

other: Reproductive NOAEC

Effect level:

7 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: 24,080 mg/m3; there were no adverse compound-related effects on reproductive function.

Remarks on result:

other: Generation: P and F1

Key result

Dose descriptor:

NOAEC

Effect level:

500 ppm (nominal)

Sex:

male/female

Remarks on result:

other: 1720 mg/m3

Remarks:

Generation: P and F1

Key result

Dose descriptor:

LOAEC

Effect level:

2 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: based on transient treatment-related sedative effect on the rats' alerting response to sound stimulus

Remarks on result:

other: 6880 mg/m3

Remarks:

Generation: P and F1

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects 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

VIABILITY (OFFSPRING)
There was a significant decrease in the mean percent born alive in high-dose F1 pups; however, this decrease was considered incidental since it was not observed in the F2 pups. There were no other changes observed in viability for F1 or F2 pups at any dose level.

CLINICAL SIGNS (OFFSPRING)
There were no treatment-related effects observed for either F1 and F2 pups.

BODY WEIGHT (OFFSPRING)
Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters (see Table 1 below).

Key result

Dose descriptor:

NOAEC

Generation:

other: F1 and F2

Effect level:

2 000 ppm

Sex:

male/female

Remarks on result:

other: 6880 mg/m3

Key result

Dose descriptor:

LOAEC

Generation:

other: F1 and F2

Effect level:

7 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: 24080 mg/m3

Reproductive effects observed:

not specified

Table 1: Mean Pup Weights (g)

Concentration (ppm)	0	500	2000	7000	0	500	2000	7000
	F1 generation				F2 generation
Day 0	6.7	6.7	6.7	6.6	6.4	6.6	6.3	6.3
Day 4 preculling	11.0	11.0	11.2	10.6	10.8	10.8	10.1	10.2
Day 4 postculling	11.0	11.0	11.3	10.6	10.9	10.8	10.1	10.1
Day 7	16.2	16.2	16.3	15.1*	16.3	16.0	15.3	14.3*
Day 14	30.0	29.9	29.7	26.5*	31.0	30.2	28.9	26.2*
Day 21	48.5	48.5	48.3	43.1*	50.0	48.3	46.4	42.8*
Day 25	67.5	67.8	68.3	62.2*	69.3	67.1	65.6	61.3*

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

Conclusions:

Decreased sound stimulus observed in 2000- and 7000-ppm animals (both sexes in both generations) was considered to be the most sensitive indicator of parental toxicity. This effect was an expected outcome of overexposure. Additional parental effects include decreased mean body weight and mean body weight gain in 7000 -ppm P and F1 rats. Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters. There were no adverse treatment regarding reproductive function.
 

Executive summary:

In a 2-generation inhalation reproduction study, cyclohexane was administered to 30 Crl:CD BR rats /sex/dose at dose levels of 0, 500, 2000, or 7000 ppm. Whole body exposures were conducted, and animals were exposed 6 hours/day, 5 days/week including holidays. For both generations, animals were exposed prior to mating, and pregnant females were exposed daily during gestation days 0 through 20; exposure cessed from gestation day 21 until lactation day 4. Exposure resumed on lactation day 5 until litters were weaned. Males continued to be exposed 5 days/week until sacrificed. Neonates were not exposed during lactation. Pups were culled on lactation day 4; however, there were no additional details provided on the culling procedure. ECD 416.

 

Decreased sound stimulus observed in 2000- and 7000 -ppm animals (both sexes in both generations) was considered to be the most sensitive indicator of parental toxicity. This effect was an expected outcome of overexposure. Additional parental effects include decreased mean body weight and mean body weight gain in 7000 -ppm P and F1 rats. Decreased male body weights observed at 7000 ppm were considered to be an artefact of body-weight deficits established as pups. Although not established by the study authors, the parental systemic LOAEC appears to be 2000 ppm (6880 mg/m3) in males and females, based on decreased sound stimulus. The parental systemic NOAEC appears to be 500 ppm (1720 mg/m3) in males and females.

 

Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters. The offspring LOAEC is 7000 ppm (24,080 mg/m3) based on decreased litter weights. The offspring NOAEC is 2000 ppm (6880 mg/m3).

There were no adverse treatment regarding reproductive function. Consequently, the reproductive NOAEC appears to be 7000 ppm (24,080 mg/m3).

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because this study was performed in accordance with GLP and appeared to closely followed OECD 416. This study will influence the DNEL.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

18 Sept 1989 - 16 June 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: (P) 28 days; (F1) 29-31 days
- Weight at study initiation: (P) Males: 75-100 g; Females: 65-80 g
- Housing: individually except during mating and lactation in stainless steel wire mesh cages, females were housed in plastic cages from gestational day (GD 20) through weaning; animals were identified by ear notches or toe clips
- Diet (e.g. ad libitum): Certified Ground Rodent Diet RMH 3200, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-73 degree F
- Humidity (%): 40-63
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark


IN-LIFE DATES: From: Sept. 18, 1989 To: June 16, 1990

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 900 l glass and stainless steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: 200 l/min
- Method of conditioning air: Test substance was metered from a piston pump into a heated glass evaporator with a temperature of 36-61 degree C. Conditioned air was passed through the evaporator, where it carried the vapor into the exposure chamber.
- Temperature, humidity: monitored every 30 minutes
- Air flow rate: 200 l/min
- Air change rate: 20 min
- Treatment of exhaust air: filtration


TEST ATMOSPHERE
- Brief description of analytical method used: GC with flame ionization detection
- Samples taken from breathing zone: yes, six times per exposure

Details on mating procedure:

- M/F ratio per cage: 1/1 - If mating failed, females were switched to the male of an unmated pair in the same dose group after 7 days. If mating failed again, they were switched after another 7 days.
- Length of cohabitation: 3 weeks, including during exposure
- Proof of pregnancy: vaginal plug, day 0

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken six times per exposure period and analyzed with GC-FID. Distribution of test substance was evaluated by sampling nine different areas of the exposure chamber.

Duration of treatment / exposure:

10 weeks pre-breeding, 3 weeks during breeding
Females continued to be exposed through GD 19. Exposure was resumed on postnatal day 5, and continued through weaning.
The F1 generation was treated similarly, but pre-breeding exposure was 8 weeks.

Frequency of treatment:

6 hrs/day, 5 days/week

Details on study schedule:

- F1 parental animals not mated until 9 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 13-16 weeks

Remarks:

Doses / Concentrations:
0, 900, 3000, 9000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
892, 2995, 9019 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

25 per sex per dose

Control animals:

yes, sham-exposed

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, toxicity, littering, mating

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: Yes, food consumption of pregnant females was measured in 3-4 day intervals, and through postnatal day 28.

Litter observations:

STANDARDISATION OF LITTERS
Parents of the F2 generation were selected on day 28 postpartum, at least one pup per litter was selected, with a second pup selected only if all litters were already represented. The F2 generation was standardized on day 4 postpartum.


PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities


GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after parturition of the first litter
- Maternal animals: All surviving animals day after weaning.


GROSS NECROPSY
- Gross necropsy consisted of external surfaces, orifices, cranial cavity, carcass, brain, spinal cord, thoracic cavity, abdominal cavity, pelvic cavity, cervical tissues and organs

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues from 25 male and females from the high dose and control groups were examined including testes of males failing to mate.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations as follows: stillborn and pups dying during lactation, culled pups


GROSS NECROPSY
- Gross necropsy consisted of external examinations.

Statistics:

Quantitative continuous variables were compared by use of Levene's test for equal variance, analysis of variance, and t-tests. Significance for t-tests were corrected by the Bonferroni method. Nonparametric data was evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney test. Indices were compared using Fisher's exact test. 0.05 was used as the criteria for statistical significance.

Reproductive indices:

mating index, fertility index, gestational index, live birth index,

Offspring viability indices:

4-day survival index, 7-day survival index, 14-day survival index, 21-day survival index, lactation index

F0 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related effects observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no treatment related effects on food consumption. Males in the 9000 ppm group had reduced body weight during week 13. Body weight gains in this group were reduced during weeks 7, 11-12, and 12-13. Males in the 3000 ppm group had reduced body weight gain in weeks 4-5, and reduced weight in weeks 9-10. Females weight gains were reduced in the 9000 ppm group in weeks 5-6.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Lactational food consumption was significantly reduced during days 7-11, and days 19-21 in the 9000 ppm group. No other reproductive parameters differed significantly from controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related abnormalities were seen.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Hyaline droplet nephropathy and tubular basophilia were seen in the 9000 ppm males.

F1 GENERATION
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related effects were observed. One female in the 900 ppm group died on day 83 due to prolonged delivery.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights of 9000 ppm males were significantly reduced throughout the exposure period. Weight gain was reduced in this group during the weeks 9-10, and 10-11. Females in the 9000 ppm group had reduced body weight during the first 3 weeks of pre-breeding exposure.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In the 9000 ppm group, food consumption was reduced on gestational days 0-4, and 4-7, and gestational intervals 0-7, and 7-14. This group also had reduced food consumption during lactational days 21-24, 26-27, 21, and 28. In the 3000 ppm group, food consumption was reduced during lactational days 22-23, and in the 900 ppm group during days lactational days 21-22.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related abnormalities were seen.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Hyaline droplet nephropathy and tubular basophilia were seen in the 9000 ppm males.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 10 560 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: reduced body weight. The value was re-calculated from the nominal dose of 3000 ppm.

Remarks on result:

other: Generation: F1, F2

Key result

Dose descriptor:

LOAEC

Effect level:

ca. 31 680 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: reduced body weight. The value was re-calculated from the nominal dose of 9000 ppm.

Remarks on result:

other: Generation: F1, F2

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 31 680 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The value was re-calculated from the nominal dose of 9000 ppm.

Remarks on result:

other: Generation: reproductive toxicity

F1 GENERATION
VIABILITY (OFFSPRING)
The number of dead pups was increased in the 900 ppm exposure group, however, as this was not seen at higher doses, it was not considered treatment related.

BODY WEIGHT (OFFSPRING)
The body weight of pups in the 9000 ppm group were reduced beginning on lactational day 14. Body weight gains in this group were reduced during lactational days 14-21 for females, and lactational days 7-14 for all pups.


GROSS PATHOLOGY (OFFSPRING)
No treatment related effects were noted.

F2 GENERATION
VIABILITY (OFFSPRING)
Viability was unaffected by exposure.

BODY WEIGHT (OFFSPRING)
The body weight of pups in the 9000 ppm group were reduced from lactational day 7-28. Body weight gains in this group were reduced during lactational days 14-21 for females, and lactational days 7-14 for all pups. There were significantly reduced body weight gains in pups in the 9000 ppm group during lactational days 4-7, and 7-14, and slightly reduced weight gains on lactational days 14-21.

GROSS PATHOLOGY (OFFSPRING)
No treatment related effects were noted.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

ca. 10 560 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

ca. 31 680 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

ca. 10 560 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

ca. 31 680 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

no

Significant Results of Reproductive Toxicity Study on Rats

Concentration (ppm)	0	900	3000	9000
Body weight of F0 adult males – week 13 (g)	463.7 (48.93)	455.2 (34.22)	455.2 (40.25)	436.1 (24.83)
Body weight gain of F0 adult males – week 4-5 (g)	32.6  (8.98)	28.9 (8.56)	24.2 (7.89)	28.9 (3.78)
Body weight gain of F0 adult males – week 6-7 (g)	25.4 (6.17)	25.4 (6.28)	23.7 (4.94)	21.2 (4.31)
Body weight gain of F0 adult males – week 9-10 (g)	24.2 (6.00)	21.6 (6.07)	18.6 (6.82)	19.9 (6.17)
Body weight gain of F0 adult males – week 11-12 (g)	11.9 (5.40)	10.7 (6.51)	12.7 (4.83)	3.3 (5.70)
Body weight gain of F0 adult males – week 12-13 (g)	11.8 (6.26)	7.4 (6.34)	8.7 (7.28)	6.4 (6.09)
Body weight gain of F0 adult females – week 0-1 (g)	0.3 (3.08)	3.4 (3.25)	1.9 (2.74)	0.8 (3.67)
Body weight gain of F0 adult females – week 5-6 (g)	11.8 (4.01)	11.0 (4.40)	12.3 (3.57)	9.0 (3.20)
Lactational food consumption F0 – day 7-11 (g/animal/day)	44.63 (3.859)	42.93 ()	43.54 (3.796)	41.45 (3.244)
Lactational food consumption F0 – day 19-21 (g/animal/day)	64.41 (5.833)	64.87 (5.439)	62.32 (6.595)	59.81 (8.212)
No. dead F1 pups - lactational day 4	5	26	12	7
F1 pup body weight – lactational day 21 (g)	41.93 (3.950)	42.50 (4.125)	39.97 (3.292)	38.92 (3.996)
F1 female pup body weight – lactational day 21 (g)	41.48 (4.151)	41.75 (4.168)	39.52 (3.430)	38.10 (4.063)
Body weight changes in F1 pups – lactational day 7-14 (g)	11.91 (1.617)	12.11 (1.328)	11.48 (1.381)	10.56 (1.780)
Body weight changes in F1 male  pups – lactational day 7-14 (g)	12.00 (1.628)	12.24 (1.306)	11.41 (1.708)	10.71 (1.847)
Body weight changes in F1 female pups – lactational day 7-14 (g)	11.81 (1.677)	12.00 (1.420)	11.51 (1.536)	10.35 (1.789)
Body weight changes in F1 female pups – lactational day 14-21 (g)	15.86 (1.933)	15.47 (2.162)	14.39 (1.744)	14.24 (2.343)
Food consumption in F1 females – week 0-1 (g/animal/day)	20.9 (1.87)	20.9 (2.00)	20.7 (2.68)	19.0 (1.62)
Food consumption in F1 females – week 1-2 (g/animal/day)	21.5 (1.45)	21.2 (2.29)	21.2 (2.80)	19.1 (1.90)
Food consumption in F1 females – week 3-4 (g/animal/day)	22.0 (2.40)	21.8 (2.74)	21.5 (2.98)	19.6 (1.99)
Food consumption in F1 females – week 5-6 (g/animal/day)	20.8 (2.02)	21.2 (2.60)	20.6 (2.87)	19.1 (2.00)
Food consumption in F1 females – week 7-8 (g/animal/day)	20.3 (1.84)	20.3 (2.24)	20.0 (2.37)	18.4 (1.99)
F1 Gestational food consumption – day 0-4 (g/animal/day)	22.87 (3.172)	21.93 (2.407)	21.93 (3.237)	19.67 (1.703)
F1 Gestational food consumption – day 4-7 (g/animal/day)	24.31 (3.047)	23.63 (3.228)	23.42 (3.077)	21.81 (2.072)
F1 Gestational food consumption – day 0-7 (g/animal/day)	23.48 (2.972)	22.44 (2.503)	22.57 (2.905)	20.56 (1.760)
F1 Gestational food consumption – day 7-14 (g/animal/day)	26.28 (3.268)	25.25 (3.108)	24.52 (3.055)	23.70 (2.565)
F1 lactational food consumption – day 21-22 (g/animal/day)	87.77 (15.326)	79.55 (8.381)	80.31 (8.272)	74.01 (9.711)
F1 lactational food consumption – day 22-23 (g/animal/day)	91.26 (10.218)	87.42 (9.649)	83.36 (8.764)	81.23 (10.532)
F1 lactational food consumption – day 23-24 (g/animal/day)	97.23 (11.339)	94.59 (9.185)	90.30 (6.703)	85.17 (13.188)
F1 lactational food consumption – day 26-27 (g/animal/day)	115.86 (11.445)	114.19 (16.261)	109.85 (11.689)	105.38 (15.023)
F1 lactational food consumption – day 21-28 (g/animal/day)	102.87 (7.787)	100.49 (8.471)	97.47 (6.852)	94.04 (10.541)

Conclusions:

The NOAEC for both male and female rats (adults and offspring) was 3000 ppm. The LOAEC for these groups was 9000 ppm based on reduced body weight. There were no adverse effects on reproduction, therefore the NOAEC for reproduction is 9000 ppm.

Executive summary:

The purpose of this study was to determine the effect of commercial hexane on reproduction in rats. Groups of 25 male and 25 female rats were exposed to nominal concentrations of 0, 900, 3000, or 9000 ppm of test substance for 10 weeks pre-breeding, 3 weeks during breeding, and postnatal days 4 -28. After weaning, pups were selected to be parents for the F2 generations, and treated similarly to their parents, except their pre-breeding exposure was 8 weeks. During exposure, animals were monitored for mortality, clinical signs, food consumption, and body weight. Offspring were examined for body weight, survival, and viability. Both parents and offspring were sacrificed and examined for gross abnormalities, and in the case of adults histopathology. Reproductive parameters were similar in exposure groups and control groups. There was reduced body weight in the F1 and F2 generation in both sexes in the 9000 ppm exposure group in both adults and offspring. The NOAEC is therefore 3000 ppm, and the LOAEC is 9000 ppm. Since there were no adverse effects in offspring without adverse maternal effects, the NOAEC for reproduction is 9000 ppm.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

The ‘justification for the read across’ is provided in the ‘Attached justification’ section below.

Species:

rat

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

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

2 key read across studies from structural analogues available for assessment.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is no substance specific reproductive data available for 2,2,4-trimethylpentane. However, data is available for structural analogues cyclohexane and hexane and presented in the dossier. This data is read across to 2,2,4-trimethylpentane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Inhalation

Cyclohexane

In a 2-generation inhalation reproduction study (Kreckmann et al., 2000), cyclohexane was administered to 30 Crl:CD BR rats /sex/dose at dose levels of 0, 500, 2000, or 7000 ppm. Whole body exposures were conducted, and animals were exposed 6 hours/day, 5 days/week including holidays. For both generations, animals were exposed prior to mating, and pregnant females were exposed daily during gestation days 0 through 20; exposure cessed from gestation day 21 until lactation day 4. Decreased sound stimulus observed in 2000- and 7000 -ppm animals (both sexes in both generations) was considered to be the most sensitive indicator of parental toxicity. This effect was an expected outcome of overexposure. Additional parental effects include decreased mean body weight and mean body weight gain in 7000 -ppm P and F1 rats. Decreased male body weights observed at 7000 ppm were considered to be an artefact of body-weight deficits established as pups. Although not established by the study authors, the parental systemic LOAEC appears to be 2000 ppm (6880 mg/m3) in males and females, based on decreased sound stimulus. The parental systemic NOAEC appears to be 500ppm (1720 mg/m3) in males and females.Mean pup weight was statistically significantly reduced from postpartum day 7 throughout the remainder of the 25-day lactation period for 7000-ppm F1 and F2 litters. The offspring LOAEC is 7000 ppm (24,080 mg/m3) based on decreased litter weights. The offspring NOAEC is 2000 ppm (6880 mg/m3).There were no adverse treatment regarding reproductive function. Consequently, the reproductive NOAEC appears to be 7000 ppm (24,080 mg/m3).

Hexane

In a reliable two generation reproduction study performed according to OECD 416, CD (Sprague Dawley) rats were whole body exposed to vapour of commercial hexane (API, 1991). Exposure to commercial hexane (approx. 52% n-hexane) was at 0, 900, 3000 or 9000 ppm for 6 h/day, 5 days/week during a 70 day (10 weeks) pre-mating period and the 21 day (3 weeks) mating period. Females were further exposed during gestation (GD 1-19) and lactation (LD 5 to weaning). The F1 generation was treated similarly but the pre-mating exposure was 8 weeks (56 days). Animal observations included mortality, clinical signs, food consumption, and body weight. Offspring were examined for body weight, survival, and viability. Both parental animals and offspring were sacrificed and examined for gross abnormalities. Histopathological examinations were conducted on adult animals.

Exposure to commercial hexane did not induce adverse effects on fertility. Reproductive indices were similar in exposed and control groups. No macroscopic or microscopic alterations in male and female reproductive organs were observed. The only significant effect was reduced body weight in the F1 and F2 generations in both sexes in the 9000 ppm exposure group both in adults and offspring.

The NOAEC for both male and female rats (adults and offspring) was 3000 ppm (corresponding to 10560 mg/m³). The LOAEC for these groups was 9000 ppm based on reduced body weight. There were no adverse effects on reproduction; therefore the NOAEC for reproduction is 9000 ppm which corresponds to 31680 mg/m³.

Additionally, in order to comply with standard information requirements for Annex X substances, an OECD 443 test is proposed for structural analogue, Hydrocarbon, C7-C9, isoalkanes (EC# 921-728-3). This read across is based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in Section 13.2 of the dossier.This endpoint will be updated subsequent to ECHA's approval of the testing proposal and availability of data upon completion of the study.

Effects on developmental toxicity

Description of key information

Cyclohexane

NOAEC for developmental toxicity in rats >7000 ppm (21000 mg/m^3).

NOAEC for developmental toxicity in rabbits >7000 ppm (21000 mg/m³).

Commercial Hexane:

NOAEC for developmental toxicity in rats = 9000 ppm (31680 mg/m³)

NOAEC for developmental toxicity in mice = 3000 ppm (10560 mg/m³)

LOAEC for developmental toxicity in mice = 9000 ppm (31680 mg/m³)

Hydrocarbons, C7-C9, isoalkanes

NOAEC for developmental toxicity in rats = 1200 ppm

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 414.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks (males) and 11 weeks (females)
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Checkers, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2 deg C
- Humidity (%): 50% +/- 10%
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

Atmospheres of cyclohexane were generated by metering the liquid test substance into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapor into the inhalation chamber air supply. The chamber concentration of cyclohexane 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.

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. Chamber-atmosphere samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed and were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization. All samples were chromatographed isothermally at 70-::C on an HP-20M Carbowax column. The chamber distribution of cyclohexane vapor was determined prior to animal exposures in the high-concentration exposure chamber and while the study was underway with animals in the low- and high-concentration chambers. The results of these determinations indicated the distribution of cyclohexane vapor was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position) for inhalation toxicology testing.

Details on mating procedure:

- Impregnation procedure: cohoused in breeding pairs (M/F ratio per cage - 1/1)
- Proof of pregnancy: vaginal plug referred to as day 0

Duration of treatment / exposure:

Assumed pregnant rats were exposed on gestation days (GD) 6-15

Frequency of treatment:

6 hrs/day

Duration of test:

GD 21

No. of animals per sex per dose:

25 pregnant females per exposure group

Control animals:

yes

Maternal examinations:

During the exposure period, animals were weighed daily and clinical signs were recorded before and after exposure. During the pre- and post-exposure periods, rats were weighed weekly and clinical signs recorded once per day. On GD 21, dams were euthanized and organs of the thoracic and abdominal cavities examined grossly.

Ovaries and uterine content:

The uterus of each animal was removed and opened. The types of implants (live and dead fetuses, and resorptions) were counted and their relative positions were recorded.

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations.

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. Litterrelated 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 (co variates: 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.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Overall mean body weight gain for the exposure period (GD 6-16) was statistically significantly reduced for female rats exposed to 7000 ppm cyclohexane (approximately 69% of control). Mean body weight gain for the exposure and post-exposure period (GD 6-21) calculated using the adjusted final body weight (GD 21 body weight minus Gravid Uterine Weight), was also statistically significantly decreased (approximately 75% of control) for female rats in the 7000 ppm group.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 2 000 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

> 7 000 ppm

Based on:

test mat.

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 dead rat fetuses. There were no statistically significant differences between control and treatment groups in early, late, or total resorptions. There were no statistically significant differences between control and treatment groups in mean fetal weight. No compound-related effect on the incidence of fetal malformations or variations was observed.

Key result

Dose descriptor:

NOAEC

Effect level:

> 7 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: no fetal effects were reported

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Conclusions:

In rats, the maternal NOAEC was 2000 ppm (6000 mg/m3), and the maternal LOAEC was 7000 ppm (21000 mg/m3) based on reduced body weight gain. The developmental NOAEC 7000 ppm (21000 mg/m3) in rats.

Executive summary:

The purpose of this study was to examine the developmental toxicity of cyclohexane in rats. Groups of 25 pregnant female rats were exposed to concentrations of 0, 500, 2000, or 7000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 21. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Maternal effects were restricted to changes in maternal body weight gain and associated with decreased food consumption. No fetal effects were reported. Maternal NOAEC was determined to be 2000 ppm (6000 mg/m3) while the fetal NOAEC was greater than the highest concentration tested.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because it is well documented and follows OECD Guideline 414.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 months
- Housing: individually in stainless steel wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rabbit Diet HF #5325, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 1 deg C
- Humidity (%): 50% +/- 10%
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

Atmospheres of cyclohexane were generated by metering the liquid test substance into a heated glass Instatherm flask with a Fluid Metering Inc. pump. Nitrogen, introduced into the flask, swept the cyclohexane vapor into the inhalation chamber air supply. The chamber concentration of cyclohexane 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.

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. Chamber-atmosphere samples were drawn by vacuum pump from representative areas of the chamber where animals were exposed and were directly injected into a Hewlett Packard model 5880 Gas Chromatograph equipped with a flame ionization. All samples were chromatographed isothermally at 70-::C on an HP-20M Carbowax column. The chamber distribution of cyclohexane vapor was determined prior to animal exposures in the high-concentration exposure chamber and while the study was underway with animals in the low- and high-concentration chambers. The results of these determinations indicated the distribution of cyclohexane vapor was sufficiently homogeneous (less than 2% difference in chamber concentration from position to position) for inhalation toxicology testing.

Details on mating procedure:

- Impregnation procedure: cohoused in breeding pairs (M/F ratio per cage - 1/1)
- Proof of pregnancy: vaginal plug referred to as day 0

Duration of treatment / exposure:

Assumed pregnant rats were exposed on gestation days (GD) 6-18

Frequency of treatment:

6 hrs/day

Duration of test:

GD 29

No. of animals per sex per dose:

20 pregnant females per exposure group

Control animals:

yes

Maternal examinations:

During the exposure period, animals were weighed daily and clinical signs were recorded before and after exposure. During the pre- and post-exposure periods, rabbits were weighed twice weekly and clinical signs recorded once per day. On GD 29, dams were euthanized and organs of the thoracic and abdominal cavities examined grossly.

Ovaries and uterine content:

The uterus of each animal was removed and opened. The types of implants (live and dead fetuses, and resorptions) were counted and their relative positions were recorded.

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations.

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. Litterrelated 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 (co variates: 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.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No treatment-related effects on maternal body weight or maternal body weight gain were observed at any interval during the study. There were no statistically significant differences in the incidence of any clinical sign observed in the does exposed to cyclohexane, compared to the control group. No compound-related effects were observed on the alerting response determined during exposure. There was a statistically significant decrease in the mean number of corpora lutea for female rabbits in the 2000 and 7000 ppm groups (8.9 and 8.8, respectively). Since ovulation and implantation occurred prior to exposure to the test substance, the decrease in the mean number of corpora lutea for females in the 2000 and 7000 ppm groups was attributed to normal biological variation. Both values were within the range of the historical controls for this facility (mean 8.9; range of 7.0- 10.9).

Key result

Dose descriptor:

NOAEC

Effect level:

> 7 000 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

> 7 000 ppm

Based on:

test mat.

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 dead rabbit fetuses. There were no statistically significant differences between control and treatment groups in early, late, or total resorptions. There were no statistically significant differences between control and treatment groups in mean fetal weight. No compound-related effect on the incidence of fetal malformations or variations was observed.

Key result

Dose descriptor:

NOAEC

Effect level:

> 7 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: no effects

Abnormalities:

not specified

Key result

Developmental effects observed:

no

Conclusions:

In rabbits, no maternal or fetal effects were reported and the NOAEC for both maternal toxicity and developmental effects were determined to be greater than the highest concentration tested.

Executive summary:

The purpose of this study was to examine the developmental toxicity of cyclohexane in rabbits. Groups of 20 pregnant female rabbits were exposed to concentrations of 0, 500, 2000, or 7000 ppm for 6 hrs/day during gestational days 6 -18. The animals were then sacrificed on GD 29. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. No maternal or fetal effects related to exposure were reported.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

September 1978 - Dezember 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study meets generally accepted scientific principles, acceptable for assessment, limited documentation.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: Food and Drug Administration 1966 "Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use", Segment II (Teratology Study).

Deviations:

yes

Remarks:

Administration via inhalation route

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CD (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Labs, Inc., Wilmington, Mass. 01887
- Age at study initiation: 9 wks
- Fasting period before study: no
- Housing: individually (except during mating)
- Diet (e.g. ad libitum): Standard Laboratory diet (Purina Lab Chow), fresh food presented as needed, except during each 6-hour exposure.
- Water (e.g. ad libitum): Automated water system (Elizabethtown Water Company), except during each 6-hour exposure.
- Acclimation period: 18 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1 cubic meter exposure chamber
- Method of holding animals in test chamber: no data
- Method of conditioning air:
- Temperature, humidity, pressure in air chamber: room temprature, dried air
- Method of particle size determination: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details given.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: no, males from in-house colony
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD6 - 15

Frequency of treatment:

6 hours/day

Duration of test:

until GD21 (all surviving dams), until day 21 postmating (all surviving non-pregnant females)

No. of animals per sex per dose:

20 females

Control animals:

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, gross signs of toxicologic or pharmacologic effects


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: GD 0, 6-15, and 21


BODY WEIGHT: Yes (including calculation of Body Weight Change)
- Time schedule for examinations: GD 0, 6-15, and 21


POST-MORTEM EXAMINATIONS: Yes, all females
- Sacrifice on gestation day # 21
- Organs examined: appendix containing the data was missing

OTHER:
Dams showing signs of abortion or premature delivery were sacrificed and fetuses obtained 19 days or later were processed and examined for skeletal anomalies. Only grossly abnormal fetuses obtained earlier than GD19 weresaved for possible future examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes (evidence of implantation, but no recognizable fetus)
- Number of late resorptions: Yes (recognizable dead fetus undergoing degeneration)
- Dead fetuses: Yes (dead fetus with no visible degeneration)
- Live fetuses

Fetal examinations:

- External examinations: Yes: all fetuses (weight, crown-rump distance from the parietal-interparietal suture to the base of the tail, malformations, sex based upon anogenital distance)
- Soft tissue examinations: Yes: two-thirds of fetuses (gross dissection and examination of viscera including internal sex determination)
- Skeletal examinations: Yes: two-thirds of fetuses (malformations and ossification variations)
- Head examinations: No data

Statistics:

Comparisons between negative and positive control and between negative control and each test substance-treated group were made where applicable (incidence data) by the chi-square method or by the F-test and Student's t-test (absolute data). When variances differed significantly, Student's t-test was appropriately modified using Cochran's approximation (t'). Mean number of live fetuses, resorptions, implantations and corpora lutea were compared to control by the one-tailed t-test.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Key result

Dose descriptor:

NOAEC

Effect level:

1 200 ppm (nominal)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEC

Effect level:

1 200 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

A) Maternal data:

Pregnancy rates were comparable between the negative control and the treated groups. No mortality occurred in the negative control and the treated groups. Mean body weight gain during the pre-dosing and the dosing intervals were comparable between negative control and treated groups, during the post-dosing interval mean weight gain was statistical significant higher in the treated groups.

Physical observations:

No indication of a treatment effect. Likewise, the data were generally comparable between the negative and the positive control groups.

Reproduction data:

Mean number of corpora lutea, implantation sites, live fetuses, resorption sites, and the incidence of dams with one or more resorption sites were comparable between the negative control and the treated groups.

Implantation efficiency values were slightly higher in the treated groups than in the negative control, in some instances differences were statistically significant, however, this was not considered indicative of an adverse effect.

In contrast, in the positive control group the mean number of live fetuses was significantly decreased and the mean number of resorption sites significantly increased compared to the negative control. Likewise, the incidence of dams with two or more resorptions was also significantly higher than in the negative control group. The mean number of corpora lutea, implantations, and the implantation efficiency value were comparable between the positive and negative control groups.

Gross postmortem examinations:

Few gross lesions were observed at necropsy of treated females (not further specified), no treatment-related effect was indicated.

B) Fetal data:

Mean fetal weights and mean crown-rump distances of both sexes were comparable for negative control and treated groups, while in the positive control group they were significantly lower. Mean numbers of male and female fetuses were comparable between negative control and treated groups. Likewise, sex ratio data was comparable for these groups. In contrast, the mean numbers of male and female fetuses in the positive control group were significantly lower compared to the negative control, due to lower numbers of fetuses in this group.

Variations in degree of ossification:

These variations may represent delays in the ossification process or slight ossification irregularities. The incidences of fetuses with ossification variations was comparable between negative control and the 400 ppm-treated group. In the 1200 ppm-treated group the incidence of fetuses with at least one ossification variation was significantly higher compared to the negative control. The incidence of litters containing fetuses with ossification variations was comparable between negative and treated groups. Likewise, the types and incidences of ossification variations were generally similar between the negative control and the treated groups.

In contrast, in the positive control group the incidence of fetuses with at least one variation was significantly higher, ossification was retarded.

Teratology data:

No treatment-related external, gross evisceration, soft tissue and skeletal malformations were observed in the fetuses of the treated and the negative control group. One late resorption from one female of the 400 ppm group showed extreme edema, however, no other unusual observations were noted in the other late resorptions of treated and negative control groups. In contrast, in the positive control group, external malformations were noted in 14.4 % of the fetuses, the most common symptom was craniorachischisis with protruding tongue and clubbed forelimbs. The incidences of soft tissue malformations were comparable between the negative control and the treated groups, no treatment-related effect was indicated. In the positive control group, these incidences were significantly higher than in the negative control.

Conclusions:

Under the design of the study the test substance, hydrocarbons, C7-C9, isoalkanes, produced no negative effects.

Executive summary:

Under the design of the study the test substance, hydrocarbons, C7-C9, isoalkanes, produced no negative effects.

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

10 560 mg/m³

Study duration:

subacute

Species:

mouse

Quality of whole database:

5 key read across studies in rodents and rabbits from structual analogues available for assessment.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There is no data available for 2,2,4-trimethylpentane. However, data is available for structural analogues, hexane, cyclohexane, and hydrocarbons, C7-C9, isoalkanes and presented in the dossier. This data is read across to 2,2,4-trimethylpentane based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

 

Cyclohexane

 

A key study (Krechmann et al., 2000) was identified on the developmental toxicity of rats and rabbits for cyclohexane. Whole body exposures were used for both rats and rabbits at concentrations of 0, 500, 2000, or 7000 ppm. 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 500 ppm (1720 mg/m³) (based upon transient sedation) or 2000 ppm (6880 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 (24080 mg/m³). For rabbits, no compound-related maternal effects were observed at concentration levels of 7000 ppm and below. Therefore the maternal NOAEC for rabbits was 7000 ppm. No compound-related evidence of developmental toxicity was observed at any test concentration. The developmental NOAEC for rabbits was 7000 ppm (24080 mg/m³), the highest concentration tested and the highest concentration permissible under national fire protection association standards.

 

Commercial Hexane

 

A key study (API, 1989) was to examine the developmental toxicity of commercial hexane in mice. Groups of 30 pregnant female mice were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6 -15. The animals were then sacrificed on GD 18. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 3000 and 9000 ppm groups. Fetuses in from dams in the 9000 ppm group had a statistically significant increase in some skeletal abnormalities. The maternal NOAEC in mice was 900 ppm (3168 mg/m³), and the LOAEC 3000 ppm based on lung color changes. The developmental NOAEC in mice was 3000 ppm (10560 mg/m³) and the LOAEC 9000 ppm (31680 mg/m³) based on skeletal abnormalities.

 

Another key study (API, 1989) was conducted to examine the developmental toxicity of commercial hexane in rats. Groups of 25 pregnant female rats were exposed to concentrations of 0, 900, 3000, or 9000 ppm for 6 hrs/day during gestational days 6-15. The animals were then sacrificed on GD 21. During the study, the animals were examined for clinical signs, mortality, food and water consumption, and body weights taken. After sacrifice, the internal organs were examined, and the uterus was examined for viable fetuses, number of resorptions, and number of corpora lutea. Fetuses were examined for malformations. Necropsy revealed color changes in the lungs of females in the 9000 ppm groups along with reduced body weight gain, and reduced food consumption. No treatment related abnormalities was seen in the fetuses. The maternal NOAEC in rats was 3000 ppm (10560 mg/m³), and the LOAEC 9000 ppm based on lung color changes, reduced body weight gain, and reduced food consumption. The developmental NOAEC in rats was 9000 ppm (31680 mg/m³).

 

Hydrocarbons, C7-C9, isoalkanes

 

A Segment II teratology study on hydrocarbons, C7-C9, isoalkanes, showed no evidence of embryonic or teratogenic effects in rats (ExxonMobil Chemical,1979). In this study, pregnant rats were exposed to 0, 400, or 1200 ppm for 6 h/day during gestational days 6 to 15. There was no mortality and no treatment-related effects to the dams. No treatment-related effects were observed in the number of live foetuses, foetal size, sex distribution, and external soft-tissue or skeletal examinations. Under the conditions of the study, there was no evidence of embryotoxicity or teratogenicity. The NOAEC for developmental toxicity was 1200 ppm, the highest dose tested.

 

Additionally, OECD Guideline 414 (Prenatal Developmental Toxicity) rodent and non-rodent species tests are proposed for structural analogue, Hydrocarbons, C7-C9, isoalkanes. This endpoint will be updated subsequent to ECHA's approval of the testing proposals and availability of data upon completion of the studies.

Justification for classification or non-classification

Based on the available read across data from structural analogues, 2,2,4-trimethylpentane does not warrant classification as a reproductive or developmental toxicant under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Additional tests (OECD 443 and OECD 414 (rodent and 2nd species)) are proposed and will be conducted on a structural analogue, Hydrocarbons, C7 -C9, isoalkanes subsequent to ECHA's approval of the same. This endpoint will be updated upon completion of the above studies subject to ECHA's approval.