Bis(methylcyclohexyl) phthalate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

2 -generation reproductive toxicity study according to OECD 416 (N. Hoshino et al., 2005)

NOAEL(males) = 240ppm (app. 16mg/kg) based on reduced anogenital distance, appearance of areola, and reduced spermatide head count in the testes.

NOAEL (females) = 1200 ppm (app. 105mg/kg) based on effects on body weight and food consumption. Female reproductive parameters were unaffected by treatment.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

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 /hr
- Photoperiod (hrs dark / hrs light): 12/12h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Pre-mating period: >= 10 weeks
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Pregnancy was confirmed by investigating the existence/absence of delivery and/or implantation sites at time of necropsy.

Duration of treatment / exposure:

- P0 females: from 5 weeks of age until necropsy through 10 weeks or more of pre-mating, mating, gestation, and lactation periods until weaning of the F1 offspring (PND 21)
- P0 males: from 5 weeks of age until necropsy through 10 weeks or more of pre-mating annd mating periods
- P1 females: from time of weaning (3 weeks of age) until necropsy through 10 weeks or more of pre-mating, mating, gestation, and lactation periods until weaning of the F2 offspring (PND 21)
- P1 males: from time of weaning (3 weeks of age) until necropsy through 10 weeks or more of pre-mating and mating periods
- Administration of non-delivering P0/P1 animals continued until necropsy, which was conducted at least 26 days after confirmation of copulation.

Frequency of treatment:

continuous

Details on study schedule:

- F1 parental animals not mated until 11-12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 14-15 weeks

Dose / conc.:

240 ppm

Remarks:

approx. 16 mg/kg (P0 males), 21 mg/kg (P0 females), 18 mg/kg (P1 males), 21 mg/kg (P1 females)

Dose / conc.:

1 200 ppm

Remarks:

approx. 80 mg/kg (P0 males), 104 mg/kg (P0 females), 90 mg/kg (P1 males), 107 mg/kg (P1 females)

Dose / conc.:

6 000 ppm

Remarks:

approx. 402 mg/kg (P0 males), 511 mg/kg (P0 females), 457 mg/kg (P1 males), 534 mg/kg (P1 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.

Parental animals: Observations and examinations:

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)

Oestrous cyclicity (parental animals):

Vaginal smears were collected everyday in the morning during the two weeks before mating, starting from 13 weeks of age for F0 parents and from 11 weeks of age for F1 parents. Estrous cycles other than 4 to 6 days were regarded as abnormal.

Sperm parameters (parental animals):

Parameters examined in 10 males from all groups of the P and F1 parental animals: sperm motility, spermtid head count in the testes, sperm count in the cauda spididymis
Parameters examined in 10 males from the high dose and control P and F1 parental animals: sperm morphology
Parameters examined in all P and F1 male animals: weight and histopathological examination of testes, epidiymes (whole and caudal), and seminal vesicles

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and stored in 10% formalin solution

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- observations and measurements: (detailed) clinical observations, number and sex of pups, stillbirths, live births, postnatal mortality, anogenital distance (PND 4), presence of nipples/areolae in male pups (PND 14 (F1), PND 12 (F2), weight gain (body weights on PNDs 0, 4, 7, 14 and 21).
- physical development and sexual maturation: incidence of pinna unfolding (from PNDs 2 to 4), day of upper incisor eruption (from PND6) and eye opening (from PND10), preputial separation (males), vaginal opening (females)

GROSS EXAMINATION OF DEAD PUPS:
yes, except pubs that died before PND 4

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Pain response, negative geotexis, air righting reflex, pinna reflex (PND 19)

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

HISTOPATHOLOGY / ORGAN WEIGTHS
- 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

On PND 21, brain, thymus, and spleen of single F1 and F2 male and female offspring were weighed.

Statistics:

Data concerning effects on the offspring until their weaning were based on values calculated per litter as the specimen unit. 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.

Reproductive indices:

Number of days required for completion of copulation, number of oestrous stages missed until completion of copulation; mating index; fertility index; gestation length; gestation index; birth index, number of implantations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1200 ppm: The body weight of mid dose females was reduced at several intervals throughout the study, but final body weight was comparable to controls. Body weight gain of males and females was inhibited.
- 6000 ppm: Significantly reduced body weight in high dose females. Body weight gain of males and females was inhibited.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption in high dose F0 females (11 - 12% in the first 2 weeks of gestation)

Compound intake (mean +/- SD):

F0 males low dose mid dose high dose
10 week period 15.88 ± 1.07 79.57 ± 3.32 401.8 ± 15.6

F0 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

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.
- Reproductive organs: focal atrophy of seminiferous tubules was noted in one each of the control and 6000 ppm groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Hormone levels (testosterone, estradiol, FSH, LH) were not affected by treatement.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle length of high dose females was significantly longer. But since the difference was small (4.25 days vs. 4 days), the value was still at the lower end of the cycle lenght considered normal (4-6 days), and the effect was not reproduced in F1 females, this finding was considered incidental.

Reproductive function: sperm measures:

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

- 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.
- No effects of the substance were apparent in the results obtained during delivery and lactation periods.
- There was no difference in the number of pups delivered, and pup viability on PNDs 0, 4, and 21.

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

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 240 ppm: One female died during delivery on GD 25. Although her delivery started on GD 22, all the pups died without suckling the milk, and the dam eventually died on GD25 before all the intrauterine fetuses were completely delivered.
- 6000 ppm: One female died on PND 26.
It was considered that the mortality was caused by systemic worsening of general condition during puerperal period or by opportunistic infection. As no similar changes were found in scheduled-sacrificed animals, it was judged that be non-treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 1200 ppm: Significantly reduced body weight in males. Body weight gain of males and females was inhibited.
- 6000 ppm: Significantly reduced body weight in males and females. Body weight gain of males and females was inhibited.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption in mid and high dose F1 males (-7.7% and - 8.5% on PND 91, respectively)

F1 males
Total study period 17.84 ± 0.86 89.89 ± 5.01 457.4 ± 17.3

F1 females
Total study period 20.95 107.15 534.2
Pre-mating 19.27 ± 1.36 98.88 ± 8.34 483.0 ± 25.7
Gestation 14.11 ± 0.98 72.41 ± 4.18 350.9 ± 19.4
Lactation 33.70 ± 2.27 170.6 ± 14.0 896.7 ± 63.2

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver weight in high dose males was significantly increased, likely as a consequence of reduced final body weight.
Absolute prostate weight was reduced in all dose groups without clear relation to dose. Weights of the low and mid dose were comparable, with the slightly higher value observed at the higher dosage. High dose animals had the lowest prostate weight (- 28%). In this group, the relative prostate weight was also significantly different from the control animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- 1200 ppm: enlarged livers in females
- 6000 ppm: enlarged livers in males and females. Soft small sized testes were observed in one high dose male. Examination of this rat revealed no sperm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Hypertrophy of hepatocytes and thyroid follicular cells in high dose animals
- Increased hyaline droplets in the renal proximal tubular epithelium accompanied with the appearance of eosinophilic microbodies in high dose males.
- 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.
- 3 animals in the 6000 ppm group showed diffuse atrophy of seminiferous tubules. Focal atrophy of the seminiferous tubules occurred in one control, two mid dose, and six high dose animals. Moreover, in the high dose group, the degree of atrophic testicular changes increased. A lack of sperm in the epidydimal tubes was observed in the three males with diffusely atrophic seminiferous tubules.

Other effects:

no effects observed

Description (incidence and severity):

Hormone levels (testosterone, estradiol, FSH, LH) were not affected by treatement.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

The number of homogenization-resistant spermatids in the testis was significantly reduced in mid and high dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

- 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.
- No effects of the substance were apparent in the results obtained during delivery and lactation periods.
- The values for the mating and fertility indices showed slight tendencies for decrease in the 6000 ppm group of the P1 parents, and this was considered associated with the testicular changes recognized in three males at necropsy. In the other males of the same group (the P1 6000 ppm group) copulation and resultant pregnancy were normal. Furthermore, no effects of the substance were apparent in the results obtained during the delivery and lactation periods.
- There was no difference in the number of pups delivered, and pup viability on PNDs 0, 4, and 21.

Dose descriptor:

NOAEL

Effect level:

240 ppm

Sex:

male

Basis for effect level:

food consumption and compound intake

reproductive function (sperm measures)

Remarks on result:

other: app. 16mg/kg

Dose descriptor:

NOAEL

Effect level:

1 200 ppm

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: app. 100 mg/kg

Critical effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

System:

male reproductive system

Organ:

testes

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no difference between the treated and the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight in high dose pups (PND 0-21) and reduced body weight gain

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

significantly reduced absolute and relative androgenital distance in high dose male pups, accompanied by the appearance of areola

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Observed changes were not consistent between groups. All changes were either incidental or secondary to reduced body weight gain.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no difference in the results of the reflex response tests, external surface anomalies, incidence of pinna unfolding, age of incisor eruption, eye opening, as well as age age and body weight for vaginal opening. The age of preputial sepearation was unaffected by treatment despite the fact that the body weight was lower in high dose pubs due to reduced body weight gain.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 200 ppm

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

Critical effects observed:

yes

Lowest effective dose / conc.:

6 000 ppm

System:

other: reduced anogenital distance in males and presence of areola

Organ:

not specified

Treatment related:

yes

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no difference between the treated and the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight in high dose pups (PND 0-21) and reduced body weight gain (PND 14-21)

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

significantly reduced absolute and relative androgenital distance in mid and high dose male pups, accompanied by the appearance of areola

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Observed changes were not consistent between groups. All changes are either incidental or secondary to reduced body weight gain.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no difference in the results of the reflex response tests, external surface anomalies, incidence of pinna unfolding, age of incisor eruption, eye opening.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

240 ppm

Sex:

male

Basis for effect level:

sexual maturation

Dose descriptor:

NOAEL

Effect level:

1 200 ppm

Sex:

female

Basis for effect level:

body weight and weight gain

Critical effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

System:

other: sexual maturation (AGD, presence of areola)

Organ:

not specified

Treatment related:

yes

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 200 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

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
P1 males
	Total study period	-	17.84 ± 0.86	89.89 ± 5.01	457.4 ± 17.3
P1 females
	Total study period	-	20.95	107.15	534.2
	Pre-mating	-	19.27 ± 1.36	98.88 ± 8.34	483.0 ± 25.7
	Gestation	-	14.11 ± 0.98	72.41 ± 4.18	350.9 ± 19.4
	Lactation	-	33.70 ± 2.27	170.6 ± 14.0	896.7 ± 63.2

^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)

 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

16 mg/kg bw/day

Species:

rat

Additional information

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

The reproductive 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, 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. 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. In the offspring, results of the reflex response tests, external surface anomalie examinations, incidence of pinna unfolding, age of incisor eruption, eye opening, preputial seperation, and vaginal opening were similar in treated and control groups.

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 specific hyaline droplets in the renal proximal tubular epithelium derived from a2µ globulin.

In the F1 animals, effects on body weight and food consumption were also detected. Here, also mid dose males were affected. Histopatholoy and organ weight results were mostly comparable, but three animals in the 6000 ppm group showed diffuse atrophy of seminiferous tubules. Focal atrophy of the seminiferous tubules occured in one control, two mid dose, and six high dose animals. Moreover, in the high dose group, the degree of atrophic testicular changes increased. A lack of sperm in the epididymal tubes was observed in the three males with diffusely atrophic seminiferous tubules. Due to these animals, mating and fertility indices were slightly reduced in this group. Additionally, the number of homogenization-resistant spermatids in the testis was significantly reduced in mid and high dose males. In the F1 high dose pups, the absolute and relative androgenital distance was reduced, accompanied by the appearance of areola.

Body weight in F2 pups was also reduced, though the difference was smaller than in the F1 generation. The relative and absolute anogenital distance was reduced in mid and high dose males, also accompanied by the presence of areola. The effect was dose dependent.

Consequently, the NOAEL in the study was 240ppm (app. 16mg/kg) for males based on reduced anogenital distance, appearance of areola, and reduced spermatide head count in the testes. For females, the NOEAL was 1200 ppm (app. 105mg/kg) based on effects on body weight and food consumption. Female reproductive parameters were unaffected by treatment.

Justification for classification or non-classification

The structurally related substance dicyclohexyl phthalate caused reduced anogenital distance and appearance of areola in male pups and, at higher concentrations in the offspring only, atrophy in the testes and reduced sperm count. These effects occured without any significant general toxicity. Consequently, the criteria for classification as toxic to reproduction (development) 1B (H360D) are fulfilled, as also stated in the harmonized classification (ATP 09). Similar effects are also expected for the registered substance bis(methylcyclohexyl)phthalate, and the same classification is applied.