Bis(methylcyclohexyl) phthalate

Endpoint summary

Description of key information

2_Gen (RA 84 -61 -7): NOAEL = 1200 ppm (Tox. Science, 2005)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

compared to standard rep. dose studies, no blood examinations and limited number of organs

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 416

Version / remarks:

1983

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

IGS (international genetic standard)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Astugi breeding center)
- Age at study initiation: (P) 5 wks; (F1) 3 wks (time of weaning)
- Housing: polycarbonate cages with bedding for lab. animals
- Diet (e.g. ad libitum): ad lib (NIH-07M, CLEA, Japan Inc.)
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 -25°C
- Humidity (%): 35-75%
- Air changes (per hr): 12/h
- Photoperiod (hrs dark / hrs light): 12/12h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): NIH-07M, CLEA, Japan Inc.

Duration of treatment / exposure:

> 90 days

Frequency of treatment:

continuous

Dose / conc.:

240 ppm

Remarks:

app. 16mg/kg (males), 21mg/kg (females)

Dose / conc.:

1 200 ppm

Remarks:

app. 80mg/kg (males), 104mg/kg (females)

Dose / conc.:

6 000 ppm

Remarks:

app. 402mg/kg (males), 511mg/kg (females)

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A preliminary dose range finding study was conducted with DCHP administered to rats at doses of 0, 600, 2000, 6000 or 20000 ppm during the period from three weeks or more pre-mating through the mating period, until necropsy for the males, and through gestation and lactation periods until postnatal day 21 (PDN 21) for the females. With regard to effects on the parental animals, in the 20000 ppm group, inhibition of body weight gain and increase in hepatic weights were observed in males and females, along with increase in adrenal weights and decrease in the weights of the thymus, spleen and ovary in the females. In the 6000 and 2000 ppm groups, increase or a tendency for increases in hepatic weights were found in both females and males, and a similar tendency was also shown in the females of the 600 ppm group. No effects on reproductive functions, delivery or lactation were found in any dose group. Regarding effects on the offspring, inhibition of body weight gain was observed in both males and females of the 20000 ppm group, and a similar trend was found in the 6000 ppm group. Based on these findings, the highest dose for the main study set at 6000 ppm, with middle and lowest doses of 1200 and 240 ppm, respectively, by dividing with the common ratio of 5.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during b.w. measurements

BODY WEIGHT: Yes
- Time schedule for examinations: weekls

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HORMONE MEASUREMENT
- Time schedule: at necropsy
- Animals: 6 females proestrous (after lactation) and 6 males per P and F1 treatment group - randomly selected
- Hormones analyzed: testosterone, FSH, LH (males), estradiol, FSH, LH (females)

ESTROUS CYCLE DETERMINATION

SPERM ANALYSIS
count, motility, morphology

Sacrifice and pathology:

All animals sacrificed or found dead

ORGAN WEIGHTS
- all animals
- brain, pituitary gland, thyroid including parathyroid, liver, kidneys, adrenal glands, spleen, testes, epididymes (whole and caudal parts), prostate (ventral lobe), seminal vesicles (including the coagulating glands), ovaries, and uterus (including the cervical region)

HISTOPATHOLOGY
- high dose and control: brain, pituitary gland, thyroid including parathyroid, liver, kidneys, adrenal glands, spleen, testes, epididymes, seminal vesicles (including coagulating glands), prostate (ventral lobe), ovaries, uterus (including the cervical region), vagina, and mammary glands
- low and mid dose: liver, thyroid, kidney (males), testes (F1 only), adrenals (F1 only)
- all macroscopically abnormal sights were additionally examined

Statistics:

Using weights of bilateral organs, the sums of the left and right organs were employed for statistical analysis.
Metric data were analyzed for homogeneity of variance by Bartlett’s method (Bartlett, 1937). When the variance was homogeneous, one-way ANOVA was carried out. When not homogeneous a Kruskal-Wallis’s test (Kruskal and Wallis, 1952) was performed. When a significant inter-group difference was found, Dunnett’s method (Dunnett, 1955) or a Dunnett type multiple-comparison method (Dunnett, 1964) were applied. For some examination items, the Kruskal-Wallis test was applied first, and when a significant inter-group difference was found, Dunnett type multiple-comparison method was conducted. Numerical data were analyzed by the Fisher’s exact probability method (Fisher, 1955). The level of statistical significance was basically set at 5%.
For clinical signs, necropsy, and histopathological findings, no statistical analyses were performed.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced body weight in high dose females. The body weight of mid dose females was reduced at several intervals throughout the study, but final body weight was comparable to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption in high dose females (11 - 12% in the first 2 weeks of gestation)

Compound intake (mean +/- SD):

males low dose mid dose high dose
10 week period 15.88 ± 1.07 79.57 ± 3.32 401.8 ± 15.6

females
Total study period 20.80 104.19 510.7
Pre-mating 17.70 ± 0.95 88.83 ± 6.39 434.6 ± 29.6
Gestation 14.30 ± 0.88 69.77 ± 4.04 349.0 ± 15.8
Lactation (days 0-21) 37.92 ± 2.50 191.6 ± 12.3 932.8 ± 58.2

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 6000 ppm: increased relative and absolute liver weight in high dose animals and increased absolute and relative thyroid weight in high dose males was noted.
- Organ weight differences were either adaptive or secondary to reduced body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

enlarged liver in high dose animals

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- diffuse hypertrophy of hepatocytes in the mid and high dose
- hypertrophy of thyroid follicular cells in mid and high dose males and high dose females
- increase in hyaline droplets in the renal proximal tubular epithelium in high dose males accompanied with the appearance of eosinophilic microbodies.

- These finding were considered not relevant, as liver hypertrophy is considered a rat-specific effect and the renal changes were concluded to be related to increased depostion of a2U globulin, which is also a rat specific effect. The thydroidal changes were considered to be linked to hypertrophy of hepatocytes.

Other effects:

no effects observed

Description (incidence and severity):

No treatmentrelated effects were observed for the sperm motility rate, number of homogenization-resistant spermatids in the testis, number of sperm in the caudal epididymis, and incidence of morphologically abnormal sperm.
Hormone levels (testosterone, estradiol, FSH, LH) were not affected by treatement.

Details on results:

Results are provided for the F0 parental generation only. For details on offspring / F1 parental generation please refer to the section on reproductive toxicology.

Dose descriptor:

NOAEL

Effect level:

1 200 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: app. 80mg/kg (males), 104mg/kg (females)

Critical effects observed:

no

Organ weight differences were either adaptive or secondary to reduced body weight. Histopathology revealed adaptive hypertrophy of hepatocytes and male rat specifichyaline droplets in the renal proximal tubular epithelium derived from a2µ globulin.

Table 1. Daily chemical intake (mg/kg)

		0 (control)	240 ppm	1200 ppm	6000 ppm
P0 males
	Total study period^b	-	15.88 ± 1.07^a	79.57 ± 3.32	401.8 ± 15.6
P0 females
	Total study period^c	-	20.8	104.19	510.7
	Pre-mating^d	-	17.70 ± 0.95	88.83 ± 6.39	434.6 ± 29.6
	Gestation^e	-	14.30 ± 0.88	69.77 ± 4.04	349.0 ± 15.8
	Lactation^f	-	37.92 ± 2.50	191.6 ± 12.3	932.8 ± 58.2

^amean ± sd

^bDuring the period of 10 weeks

^cThe mean daily intake fort he study (pre-mating + gestation + lactation periods).

^dPre-mating period (10 weeks)

^eGestation length (Days 21-23)

^fLactation period (Days 0-21)

Endpoint conclusion

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

Bis(methylcyclohexyl)phthalate was not tested in a repeated dose study. Instead, data are adapted from the structurally similar dicyclohexyl phthalate (DCHP). A detailled read across justification has been attached to section 13.

The toxicity of DCHP was evaluated in a two generation test according to OECD 416, in which male and female Sprague-Dawley (SD) rats of parental (F0) and F1 generation were exposed to DCHP in the diet at concentrations of 0 (control), 240, 1200 or 6000 ppm for more than 90 days, including a premating period of at least 10 weeks. Examinations included histopathology of several organs, detailled sperm analysis, estrous cycle length, hormone analysis, sexual maturation, and reproductive indices, but lacked further analysis of blood or urine parameters. No significant differences were observed in any dose groups for the following indices: number of days required for completion of copulation, number of estrous stages missed until completion of copulation, mating index, fertility index, gestation length, gestation index, birth index and number of implantations. Adverse effects in the high dose F0 animals were limited to reduced body weight (gain) and reduced food consumption (app - 8%). Organ weight differences were either adaptive or secondary to reduced body weight. Histopathology revealed adaptive hypertrophy of hepatocytes and male rat specifichyaline droplets in the renal proximal tubular epithelium derived from a2µ globulin.

Consequently, the NOAEL was 1200 ppm, which equals app. 80mg/kg in males and 104mg/kg in females, based on effects on body weight and food consumption.

Data were only provided for the F0 parental generation. For details on offspring and F1 parental animals, please refer to the section on reproductive toxicity.

Justification for classification or non-classification

Repeated exposure for more than 90 -days with the read across substance DCHP did not cause adverse effects up to 80 - 100mg/kg. Even at app. 500mg/kg, effects were limited to general signs of toxicity, i.e., reduced body weight and food consumption. Similar effects are also expected for the registered substance bis(methylcyclohexyl)phthalate. No classification is required according to EU GHS for STOT RE.