Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline Study in Japanese with English translation. Based on a read across evaluation, the test substance has a comparable physicochemical profile and (slightly) higher toxicological profile, therefore it is considered as a worst case source chemical for read across. Based on this conservative viewpoint, the data are considered relevant, reliable and adequate for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc
- Age at study initiation: (P) Males & Females :8 weeks
- Weight at study initiation: (P) Males: 309-337 g ; Females: 181-225 g
- Housing: Polycarbonate cages with experimental animal beddings (Beta Chip, Charles River Japan, Inc.) individually after administration, in a pair of male and female during mating period, or with a litter during lactation period in one cage.
- Diet: Ad libitum; autoclave-sterilized solid diet for experimental animals (CRF-1, Oriental Yeast Co).
- Water: Filter and irradiated with UV light ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Based on a 10-day dose range finding study, the dose of 1000 mg/kg was set as the highest dose, with 200 mg/kg and 40 mg/kg for the middle and low doses, respectively, with a common ratio of 5.
Administration period included 14 days prior to mating and mating period in both sexes, more specifically a total of 43 days until the day before scheduled sacrifice in males and from the day of successful mating, followed by delivery until post-partum day 3 in females. The test substance, which was dissolved in an olive oil, was gavaged once a day in the morning using a gastric tube. Administration volume was set to 5 mL/kg and determined based on the latest measured body weight.

Details on mating procedure:

After a 14-day of pre-mating administration period, males and females were cohabited day and night on a one-to-one basis within the same group for up to 7 days. Vaginal smears were collected every morning and examined microscopically after staining with Giemsa. Copulation was confirmed by finding of vaginal plugs or sperm in vaginal smears, with that day designated as day 0 of gestation. Males and females of the mated pairs were separated and used for the examination thereafter. Based on these results, pairing days until mating (day required for copulation after cohabitation), number of oestrous stages without mating, mating index ([number of pairs with successful mating/number of pairs examined]×100) and fertility index ([number of pregnant animals/number of pairs with successful mating]×100) were obtained.

Duration of treatment / exposure:

Males: 43 days
Females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0 (Vehicle), 40, 200, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

For the females with confirmed copulation, all of them were allowed to deliver naturally to observe the conditions of parturition. When parturition is completed by 9:00 a.m., the dams were considered to have delivered on that day and that day was designated as day 0 post partum. Then, dams were observed for clinical signs and maternal behaviour including lactation, nest building and cannibalism on a daily basis until the neonates become 4 days old (day 4 post partum).
At necropsy of day 4 post partum, the ovaries and uterus were removed and examined for the numbers of corpora lutea and implantations. Animals that had no parturition were necropsied 25 days after copulation and uteri without visible implantation sites were immersed in 2 % KOH solution to facilitate the detection of implantation sites. Based on these results, gestation period (period from day 0 of pregnancy to the day that parturition was confirmed), gestation index ([number of females with live offspring/number of pregnant females]×100), implantation index([number of implantation sites/number of corpora]×100), delivery index ([number of offspring delivered/number of offspring implantation sites]×100) were obtained.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed daily for survival, external anomalies and behaviours. Animals that died were necropsied promptly upon discovery.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
Body weight was measured at the beginning of the administration and once a week thereafter for males and at the beginning of the administration, once weekly before mating and on days 0 (day of copulation), 7, 14 and 20 of gestation and days 0 (day of parturition) and 4 post partum for mated females.

FOOD CONSUMPTION: Yes
Food consumption was measured on the same days that body weights were measured except during mating period.

OTHER:
HAEMATOLOGYY: Yes in male rats
All surviving male rats were fasted approximately 21 hours before sacrifice and blood was collected from posterior aorta under anaesthesia with the intraperitoneal administration of thiopental sodium. After treating some of the blood with an anti-coagulator, EDTA-2K, RBC , WBC, platelet count, haemoglobin concentration and haematocrit value using a multi-channel auto-analyzer (NE-4500: Toa Medical Electronics Inc), WBC percentage (Wright's stained blood smears) using a blood cell auto-analyzer (MICROX HEG-70A: Tateichi Electronics) and reticulocyte count (Argon Laser Flow cytometry method) using a reticulocyte auto-analyzer (R-2000: Toa Medical electronics Inc.), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCV) and mean corpuscular haemoglobin concentration (MCHC) were examinded.

CLINICAL CHEMISTRY: Yes in male rats
Blood was collected from all surviving male rats on the day of sacrifice. After incubating at room temperature for approximately 30 minutes, the blood was centrifuged at 3000 rpm for 10 minutes to obtain serum. The serum was analyzed for GOT, GPT, ALP , gamma-GTP, urea nitrogen, glucose, total cholesterol, triglyceride, creatinine, total bilirubin, total protein, albumin, A/G ratio, calcium, inorganic phosphate, sodium, potassium, chloride using an autoanalyzer (Hitachi 736-10 model: Hitachi Inc.).

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight (absolute and relative), epididymis weight (absolute and relative).
other: Pre-coital days, Coital rate, (Pregnancy Rate) = Reproductive performance parameters (Table 19 on p.53 of AMS Reprotox Report pdf)

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
- In vivo: number and sex of pups, stillbirths, live births, postnatal mortality, body weight and weight gain
- Postmortem: gross examination of pups

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 44
- Maternal animals: All surviving animals on day 4 of lactation

GROSS PATHOLOGY & ORGAN WEIGHTS: Yes
- Both surviving female and male rats were sacrificed by exsanguination from the abdominal aorta under anaesthesia with the intraperitoneal administration of thiopental sodium on the next day of the last day of administration.
- Weights of the thymus, liver, kidneys, testes and epididymides were measured.
- In addition to these organs, the brain, heart, spleen, adrenals, ovaries, and urinary bladder of the males with abnormal necropsy findings were collected and stored after fixing with 10 % PBS neutral buffered formalin solution (testes and epididymides were fixed with Bouin's solution).

HISTOPATHOLOGY: Yes
- Haematoxylin and eosin stained specimens were conventionally prepared for the brain, heart, liver, spleen, kidneys, adrenals, testes and epididymides of the control group and the 1000 mg/kg group in both sexes, and for the urinary bladders of both sexes and for the thymus of female for the rats with suspected influence of the test substances based on the necropsy findings and the results of organ weight measurement, and they were analyzed microscopically. As a result, since there were changes attributable to the test substance observed in the liver, kidney and adrenals of both sexes, the urinary bladder of males and the thymus of females, the same organs of the rats in the 200 and 40 mg/kg groups were examined.
- For the male rat that died in the 1000 mg/kg group before the end of the study, seminal vesicles, prostate grand, lung, stomach, duodenum and urinary duct were examined in addition to the above organs. Furthermore, the ovaries of the non-pregnant female and the skin of the rat in the 1000 mg/kg group in which crust was observed in necropsy findings were examined as well. In addition, some of the liver, kidneys and adrenals were stained with oil red O.

Postmortem examinations (offspring):

After examining for external anomalies, all surviving offspring were sacrificed by exsanguination from the abdominal aorta under anaesthesia with the intraperitoneal administration of thiopental sodium.

Statistics:

Metrical data were tested for homogeneity of variance by Bartlett’s test. When the variance was homogeneous, the one-way analysis of variance was performed, and when heterogeneous, the Kruskal-Wallis test was used. When a significant inter-group difference was found and if the number of samples in each group are the same, Dunnett’s test or the Dunnett-type multiple comparison test was employed, and if not, Scheffe test or the Scheffe type multiple comparison test was employed. However, Kruskal-Wallis test was used for the following items with *. The numerical data were analyzed with Fisher’s exact probability test. The level of significance was set at 5 % for all analyses. For the data pertaining neonates, a mean value calculated for each dam was used as a statistical unit. The following items were statistically analyzed.

Multiple comparison tests
Bodyweight, food consumption, haematological examination ,blood chemical examination, organ weight, pairing days until mating *, number of oestrous stages without mating *, gestation period *, number of corpora lutea, number of implantation sites, implantation index*, delivery index*, number of offspring delivered, live birth index* and viability index on day 4*.

Fisher’s exact probability test
Mating index, fertility index, gestation index and sex ratio (male /female)

Reproductive indices:

Gestation, Implantation and Delivery

Offspring viability indices:

Live birth index ([number of live offspring day 0/number of offspring delivered]×100) and day 4 viability index([number of live offspring on day 4/number of live offspring on day 0]×100) were obtained based on the live offspring at days 0 and 4 post partum.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation in males (all dose levels) and females (highest dose)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased in the 1000 mg/kg male group and slight suppression in 1000 mg/kg female groups during late gestation period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased in the 1000 mg/kg male group and slight suppression in 1000 mg/kg female groups during late gestation period

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver (acidophilic), adrenals (fatty droplets), kidney (hyaline droplets and basophilic change), urinary bladder (hyperplasia of the mucosal epithelium, vacuolation and infiltration), thymus atrophy in male and/or females dosed at (200 and) 1000 mg/kg

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 1000 mg/kg group, male rats one animal died due to ischuria with urinary calculi.
Salivation was observed in males (all dose groups with dose related increased incidence) and females (sporadically in mid but mainly in high dose group).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the 1000 mg/kg group, male rats showed suppression of body weight gain. Female rats of 1000 mg/kg group showed a slight suppression of body weight gain in the late gestation period.

HAEMATOLOGY
No effects

CLINICAL CHEMISTRY
In the 1000 mg/kg group blood chemical examination in male rats showed increases in GPT, urea nitrogen and potassium, and a decrease in triglyceride. In the 200 mg/kg group, an increase in GPT was observed in male rats.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Not examined (gavage study)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- The compound had no effects on reproductive parameters such as the mating index, the fertility index, gestation length, number of corpora lutea or implantations, the implantation index, the gestation index, the delivery index or parturition. In addition, for the dams that did not delivered, necropsy findings indicated a trace of plantation in the ovaries.
- Although one female rat and two female rats in the 200 mg/kg group and the 1000 mg/kg group, respectively were found to not to be pregnant, there were no significant differences between the control group and test substance treatment group in terms of the mating index and the fertility index. In addition, almost all females in each group copulated during the initial oestrous stage within four days after the initiation of mating, and there were no significant differences in the number of days until copulation and the number of oestrous stages without copulation between the control group and the test substance treatment groups. In addition, necropsy of one female in the control group revealed pregnancy although the earlier vaginal smear test did not indicate copulation.
- Regarding the observation of lactation period, total litter loss was observed in two dams in the 1000 mg/kg group. One of the dams was not lactating well to neonates and she cannibalized all offspring on the following day. In addition, the other dam did not show abnormal lactation behaviour on the day of delivery and she was lactating. But vaginal bleeding was observed on day 1 post partum when total litter was lost, the areas near the vagina and anus were dirty on the next day and hypolocomotive activity, skinniness and piloerection were observed on day 4 post partum. Furthermore, one of these two dams had significant suppression of body weight gain during the late gestation period and the other dam also showed significant loss of body weight during the lactation period. There were no anomalies observed in other dams.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increases of absolute organ weight and organ weight relative to body weight of the liver were observed in males of the 1000 mg/kg groups and females at >= 200 mg/kg. Furthermore, increases of absolute organ weight and organ weight relative to body weight of the kidneys were observed in both sexes of the 1000 mg/kg group and the increase of organ weight relative to body weight was observed in females of the 200 mg/kg group. In addition, decrease of absolute organ weight and organ weight relative to body weight of the thymus was observed in female of the 1000 mg/kg group. In addition, despite the high value of organ weight relative to body weight of the testes in the 1000 mg/kg group, it was judged to be a superficial change reflecting the suppression of body weight gain because there were no significant differences in absolute organ weight compared to the control group and there were no changes attributable to the test substance in histological and reproductive performance studies.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Liver enlargement was observed in both sexes of the 1000 mg/kg group and dark reddish change was observed only in males at >= 200 mg/kg.
- Kidney enlargement was observed in both sexes of the 1000 mg/kg group and discoloration of the cortico-medullary junction was observed only in females at >= 200 mg/kg. In addition, although there was one female case of enlargement in the 40 mg/kg group, it was judged to be an incidental lesion since the occurrence was unilateral.
- In the urinary bladder, yellow micro-granular calculi was observed only in male of the 1000 mg/kg group.
- Enlargement and grayish change in the adrenals and atrophy in the thymus were individually observed in females of the 1000 mg/kg group. The female rat, that showed thymus atrophy, also showed spleen atrophy.
- In addition, despite a case of unilateral atrophy in the testes in a male of the control group and another case of skin crust in a male of the 1000 mg/kg group, they were judged to be incidental lesions due to the circumstances of occurrence.
-For the male rat died in the 1000 mg/kg group, a large amount of blood-like discoloration of urine was found in the distended urinary bladder and 7-8 urinary calculi with 1-2 mm in diameter were observed. Enlarged kidneys, distinct bleeding in renal pelvis, urinary ducts, urinary bladder and epididymides and prostate grand adjacent to the urinary bladder were found, indicating ischuria. In addition, pulmonary oedema and spleen atrophy were observed.


HISTOPATHOLOGY (PARENTAL ANIMALS)
In the 1000 mg/kg/day group, histopathological examination demonstrated acidophilic change of hepatocytes and increase of fatty droplets in the fascicular zone of the adrenals in both sexes, increase of hyaline droplets and basophilic change in the renal tubular epithelium and hyperplasia of the mucosal epithelium in the urinary bladder in male rats, and vacuolation and infiltration of lymphocytes in the renal tubular epithelium and atrophy of the thymus in female rats.
In the 200 mg/kg group, similar histopathological changes were found in the liver and kidneys of both sexes, and the thymus of female rats.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slight decrease of body weight at 1000 mg/kg

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Although there were no significant differences between the control group and the dosed groups, the day 4 viability index slightly decreased because all neonates of two litters in the 1000 mg/kg group died. However, there were no postnatal deaths except for the above. There were no significant differences between the control group and the test substance treatment group in the number of offspring born, the number of live offspring, the sex ratio, or birth rate.

CLINICAL SIGNS (OFFSPRING)
No abnormal findings ascribable to the compound were found for external examination, clinical signs of the offspring.

BODY WEIGHT (OFFSPRING)
On examination of neonates, the 1000 mg/kg/day group showed a slight decrease of body weight. There were no significant differences in body weight gain after birth.

GROSS PATHOLOGY (OFFSPRING)
No abnormal findings ascribable to the compound were found for external examination or necropsy of the offspring.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and lower viability index due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, or body weight gain after birth. No abnormal findings ascribable to the compound were found for external examination, clinical signs, or necropsy of the offspring.

Conclusions:

The NOELs for reproductive (and developmental) toxicity are considered over 1000 mg/kg/day for parental males, and 200 mg/kg/day for parental females and offspring.

Executive summary:

1-Methylethenylbenzene was studied in SD (Crj:CD) rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 40, 200, and 1000 mg/kg/day by oral administration for 43 days from 14 days prior to mating to days after mating in males and for the periods including 14 days prior to mating and from gestation period, followed by delivery until post-partum day 3 in females.

The test substance had no effects on reproductive parameters such as the mating index, the fertility index, gestation period, number of corpora lutea or implantations, the implantation index, the gestation index, the delivery index or parturition. Two dams of the 1000 mg/kg/day group, however, lost all their offspring dung the lactation period.

Parental toxicity was observed at 1000 mg/kg bw, as one male rat died due to ischuria with urinary calculi, and increases in GPT, BUN and potassium, and decrease in triglyceride were observed in this group. Gross pathological changes were observed in various organs at 1000 mg/kg bw, confirmed to be target organ changes by histopathological examination: liver (acidophilic changes), kidney (hyaline degeneration of tubular epithelium of kidneys and fatty droplets in adrenal cortex), urinary bladder (epithelial hyperplasia in males), and thymus (atrophy in female rats). In the 200 mg/kg/day group, an increase in serum GPT and similar histopathological changes were observed in the liver, kidneys and thymus. Lowest observed effect level for systemic toxicity in parents was therefore considered to be 200 mg/kg bw.

On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and a slightly low viability index on post-partum day 4 due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, external examination, clinical signs, body weight gain after birth, or necropsy of the offspring.

The NOELs for reproductive and developmental toxicity were 1000 mg/kg/day for parental males and 200 mg/kg/day for parental females and offspring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity data were present for Depanol I, nor were there any found for source chemical d-limonene. Therefore another (worst case) source chemical was used as read across substance: 1-Methylethenylbenzene or alpha-methylstyrene. Justification for read across based on the ECHA guidance is provided in Section 13.

1-Methylethenylbenzene was studied in SD (Crj:CD) rats in an OECD 422 combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 40, 200, and 1000 mg/kg/day by oral administration for 43 days from 14 days prior to mating to days after mating in males and for the periods including 14 days prior to mating and from gestation period, followed by delivery until post-partum day 3 in females.

The test substance had no effects on reproductive parameters such as the mating index, the fertility index, gestation period, number of corpora lutea or implantations, the implantation index, the gestation index, the delivery index or parturition. Two dams of the 1000 mg/kg/day group, however, lost all their offspring during the lactation period. Parental toxicity was observed at 1000 mg/kg bw, as one male rat died due to ischuria with urinary calculi and increases in GPT, BUN and potassium, and decrease in triglyceride, were observed in this group. Gross pathological changes were observed in various organs at 1000 mg/kg bw, confirmed to be target organ changes by histopathological examination in: liver (acidophilic changes), kidney (hyaline degeneration of tubular epithelium of kidneys and fatty droplets in adrenal cortex), urinary bladder (epithelial hyperplasia in males), and thymus (atrophy in female rats). In the 200 mg/kg/day group, an increase in serum GPT and similar histopathological changes were observed in the liver, kidneys and thymus. Lowest observed effect level for systemic toxicity in parents was therefore considered to be 200 mg/kg bw.

On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and a slightly low viability index on post-partum day 4 due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, external examination, clinical signs, body weight gain after birth, or necropsy of the offspring.

The NOELs for reproductive and developmental toxicity were 1000 mg/kg/day for parental males and 200 mg/kg/day for parental females and offspring.

For this 'worst case' source chemical, slight systemic toxicity was observed at 200 mg/kg bw, however without reproductive toxicity. At the dose of 1000 mg/kg, toxicity was more extensive, most probably with impact on the prenatal and neonatal development. Therefore the loss of offspring in two dams and lower body weight and viability of pups at 1000 mg/kg bw were considered to be secondary to maternal systemic toxicity.

Short description of key information:
Read across from source chemical 1-Methylethenylbenzene or alpha-methylstyrene tested in an OECD 422 study, demonstrated systemic toxicity at 200 mg/kg bw, without reproductive toxicity. At the dose of 1000 mg/kg, maternal toxicity was more extensive, and loss of offspring in two dams and lower body weight and viability of pups were observed at 1000 mg/kg bw. These effects however were considered to be secondary to maternal systemic toxicity.

Justification for selection of Effect on fertility via oral route:
The combined repeated dose reproductive and developmental toxicity study for alpha-methylstyrene was selected based on the available data from source chemicals.

Effects on developmental toxicity

Description of key information

No teratogenicity was observed for source chemicals alpha-methylstyrene (in rats) and for d-limonene (in rats, mice and rabbits). Some foetal toxicity was observed with both source chemicals at maternally toxic doses in rats, which was considered a secondary effect, however in mice and rabbits there was no foetal toxicity even at maternally toxic doses. Developmental NOAEL was at least 591 mg/kg bw. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline Study in Japanese with English translation. Based on a read across evaluation, the registered substance has a comparable physicochemical profile and toxicological profile to alfa methylstyrene; alfa-methylstyrene can be considered as a worst case source chemical for read across. Based on this conservative viewpoint, the data are considered relevant, reliable and adequate for classification.

Qualifier:

according to guideline

Guideline:

other: OECD 422

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc
- Age at study initiation:(P) Males & Females :8 weeks
- Weight at study initiation: (P) Males: 309-337 g ; Females:181-225 g
- Housing: Polycarbonate cages with experimental animal beddings (Beta Chip, Charles River Japan, Inc.) individually after administration, in a pair of male and female during mating period, or with a litter during lactation period in one cage.
- Diet: Ad libitum; autoclave-sterilized solid diet for experimental animals (CRF-1, Oriental Yeast Co).
- Water: Filter and irradiated with UV light ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C
- Humidity: 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

Based on a 10-day dose range finding study, the dose of 1000 mg/kg was set as the highest dose, with 200 mg/kg and 40 mg/kg for the middle and low doses, respectively, with a common ratio of 5.
Administration period included 14 days prior to mating and mating period in both sexes, more specifically a total of 43 days until the day before scheduled sacrifice in males and from the day of successful mating, followed by delivery until post-partum day 3 in females. The test substance, which was dissolved in an olive oil, was gavaged once a day in the morning using a gastric tube. Administration volume was set to 5 mL/kg and determined based on the latest measured body weight.

Details on mating procedure:

After a 14-day of pre-mating administration period, males and females were cohabited day and night on a one-to-one basis within the same group for up to 7 days. Vaginal smears were collected every morning and examined microscopically after staining with Giemsa. Copulation was confirmed by finding of vaginal plugs or sperm in vaginal smears, with that day designated as day 0 of gestation. Males and females of the mated pairs were separated and used for the examination thereafter. Based on these results, pairing days until mating (day required for copulation after cohabitation), number of oestrous stages without mating, mating index ([number of pairs with successful mating/number of pairs examined]×100) and fertility index ([number of pregnant animals/number of pairs with successful mating]×100) were obtained.

Duration of treatment / exposure:

Males: 43 days
Females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

Once daily

Duration of test:

Males: 43 days
Females: from 14 days before mating to day 3 of lactation

Remarks:

Doses / Concentrations:
0 (Vehicle), 40, 200, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

For the females with confirmed copulation, all of them were allowed to deliver naturally to observe the conditions of parturition. When parturition is completed by 9:00 a.m., the dams were considered to have delivered on that day and that day was designated as day 0 post partum. Then, dams were observed for clinical signs and maternal behaviour including lactation, nest building and cannibalism on a daily basis until the neonates become 4 days old (day 4 post partum).
At necropsy of day 4 post partum, the ovaries and uterus were removed and examined for the numbers of corpora lutea and implantations. Animals that had no parturition were necropsied 25 days after copulation and uteri without visible implantation sites were immersed in 2 % KOH solution to facilitate the detection of implantation sites. Based on these results, gestation period (period from day 0 of pregnancy to the day that parturition was confirmed), gestation index ([number of females with live offspring/number of pregnant females]×100), implantation index([number of implantation sites/number of corpora]×100), delivery index ([number of offspring delivered/number of offspring implantation sites]×100) were obtained.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed daily for survival, external anomalies and behaviours. Animals that died were necropsied promptly upon discovery.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
Body weight was measured at the beginning of the administration and once a week thereafter for males and at the beginning of the administration, once weekly before mating and on days 0 (day of copulation), 7, 14 and 20 of gestation and days 0 (day of parturition) and 4 post partum for mated females.

FOOD CONSUMPTION: Yes
Food consumption was measured on the same days that body weights were measured except during mating period.

OTHER:
HAEMATOLOGYY: Yes in male rats
All surviving male rats were fasted approximately 21 hours before sacrifice and blood was collected from posterior aorta under anaesthesia with the intraperitoneal administration of thiopental sodium. After treating some of the blood with an anti-coagulator, EDTA-2K, RBC , WBC, platelet count, haemoglobin concentration and haematocrit value using a multi-channel auto-analyzer (NE-4500: Toa Medical Electronics Inc), WBC percentage (Wright's stained blood smears) using a blood cell auto-analyzer (MICROX HEG-70A: Tateichi Electronics) and reticulocyte count (Argon Laser Flow cytometry method) using a reticulocyte auto-analyzer (R-2000: Toa Medical electronics Inc.), mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCV) and mean corpuscular haemoglobin concentration (MCHC) were examinded.

CLINICAL CHEMISTRY: Yes in male rats
Blood was collected from all surviving male rats on the day of sacrifice. After incubating at room temperature for approximately 30 minutes, the blood was centrifuged at 3000 rpm for 10 minutes to obtain serum. The serum was analyzed for GOT, GPT, ALP , gamma-GTP, urea nitrogen, glucose, total cholesterol, triglyceride, creatinine, total bilirubin, total protein, albumin, A/G ratio, calcium, inorganic phosphate, sodium, potassium, chloride using an autoanalyzer (Hitachi 736-10 model: Hitachi Inc.).

SACRIFICE
- Male animals: All surviving animals on day 44
- Maternal animals: All surviving animals on day 4 of lactation

GROSS PATHOLOGY & ORGAN WEIGHTS: Yes
- Both surviving female and male rats were sacrificed by exsanguination from the abdominal aorta under anaesthesia with the intraperitoneal administration of thiopental sodium on the next day of the last day of administration.
- Weights of the thymus, liver, kidneys, testes and epididymides were measured.
- In addition to these organs, the brain, heart, spleen, adrenals, ovaries, and urinary bladder of the males with abnormal necropsy findings were collected and stored after fixing with 10 % PBS neutral buffered formalin solution (testes and epididymides were fixed with Bouin's solution).

HISTOPATHOLOGY: Yes
- Haematoxylin and eosin stained specimens were conventionally prepared for the brain, heart, liver, spleen, kidneys, adrenals, testes and epididymides of the control group and the 1000 mg/kg group in both sexes, and for the urinary bladders of both sexes and for the thymus of female for the rats with suspected influence of the test substances based on the necropsy findings and the results of organ weight measurement, and they were analyzed microscopically. As a result, since there were changes attributable to the test substance observed in the liver, kidney and adrenals of both sexes, the urinary bladder of males and the thymus of females, the same organs of the rats in the 200 and 40 mg/kg groups were examined.
- For the male rat that died in the 1000 mg/kg group before the end of the study, seminal vesicles, prostate grand, lung, stomach, duodenum and urinary duct were examined in addition to the above organs. Furthermore, the ovaries of the non-pregnant female and the skin of the rat in the 1000 mg/kg group in which crust was observed in necropsy findings were examined as well. In addition, some of the liver, kidneys and adrenals were stained with oil red O.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes , terminal kill on day 4 of lactation
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Body weight and body weight gain of F1 males and females were recorded.On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and low viability index due to the total litter losses of the two dams.
No abnormal findings ascribable to the compound were found for external examination, clinical signs, or necropsy of the offspring.

Statistics:

Metrical data were tested for homogeneity of variance by Bartlett’s test. When the variance was homogeneous, the one-way analysis of variance was performed, and when heterogeneous, the Kruskal-Wallis test was used. When a significant inter-group difference was found and if the number of samples in each group are the same, Dunnett’s test or the Dunnett-type multiple comparison test was employed, and if not, Scheffe test or the Scheffe type multiple comparison test was employed. However, Kruskal-Wallis test was used for the following items with *. The numerical data were analyzed with Fisher’s exact probability test. The level of significance was set at 5 % for all analyses. For the data pertaining neonates, a mean value calculated for each dam was used as a statistical unit. The following items were statistically analyzed.

Multiple comparison tests
Bodyweight, food consumption, haematological examination ,blood chemical examination, organ weight, pairing days until mating *, number of oestrous stages without mating *, gestation period *, number of corpora lutea, number of implantation sites, implantation index*, delivery index*, number of offspring delivered, live birth index* and viability index on day 4*.

Fisher’s exact probability test
Mating index, fertility index, gestation index and sex ratio (male /female)

Indices:

Indices related to offspring:
- Total No. of offspring at birth
- No. of live offspring at birth
- No. of live offspring on day 4
- Delivery index
- Viability index

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Female rats of 1000 mg/kg group showed a slight suppression of body weight gain in the late gestation period. In the 1000 mg/kg/day group, histopathological examination demonstrated acidophilic change of hepatocytes and increase of fatty droplets in the fascicular zone of the adrenals in both sexes and vacuolation and infiltration of lymphocytes in the renal tubular epithelium and atrophy of the thymus in female rats.
In the 200 mg/kg group, similar histopathological changes were found in the liver and kidneys of both sexes, and the thymus of female rats.

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: embryotoxicity

Details on embryotoxic / teratogenic effects:
Two dams of the 1000 mg/kg/day group lost all their pups dung the lactation period.
On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and low viability index due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, or body
weight gain after birth. No abnormal findings ascribable to the compound were found for external examination, clinical signs, or necropsy of the offspring.

Abnormalities:

not specified

Developmental effects observed:

not specified

On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and low viability index due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, or body weight gain after birth. No abnormal findings ascribable to the compound were found for external examination, clinical signs, or necropsy of the offspring.

Conclusions:

The NOELs for developmental toxicity was 200 mg/kg/day for parental females and offspring.

Executive summary:

1-Methylethenylbenzene was studied in SD (Crj:CD) rats in an OECD 422 combined repeated dose and reproductive/developmental toxicity screening test at doses of 0, 40, 200, and 1000 mg/kg/dayby oral administration for 43 days from 14 days prior to mating to days after mating in males and for the periods including 14 days prior to mating and from gestation period, followed by delivery until post-partum day 3 in females.

The test substance had no effects on reproductive parameters such as the mating index, the fertility index, gestation period, number of corpora lutea or implantations, the implantation index, the gestation index, the delivery index or parturition. Two dams of the 1000 mg/kg/day group, however, lost all their offspring dung the lactation period.

Parental toxicity was observed at 1000 mg/kg bw, as one male rat died due to ischuria with urinary calculi, and increases in GPT, BUN and potassium, and decrease in triglyceride were observed in this group. Gross pathological changes were observed in various organs at 1000 mg/kg bw, confirmed to be target organ changes by histopathological examination: liver (acidophilic changes), kidney (hyaline degeneration of tubular epithelium of kidneys and fatty droplets in adrenal cortex), urinary bladder (epithelial hyperplasia in males), and thymus (atrophy in female rats). In the 200 mg/kg/day group, an increase in serum GPT and similar histopathological changes were observed in the liver, kidneys and thymus. Lowest observed effect level for systemic toxicity in parents was therefore considered to be 200 mg/kg bw.

On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and a slightly low viability index on post-partum day 4 due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, external examination, clinical signs, body weight gain after birth, or necropsy of the offspring.

The NOELs for reproductive and developmental toxicity were 1000 mg/kg/day for parental males and 200 mg/kg/day for parental females and offspring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

591 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Various studies were available, which in combination, were considered of high quality.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No developmental toxicity studies were available for Depanol I, however there were various studies available from source chemicals Alpha-methylstyrene in rats and from d-limonene in rats, rabbits and mice. Justification for read across based on the ECHA guidance is provided in Section 13.

All these studies demonstrated absence of (primary) developmental toxicity and absence of teratogenicity. The information was considered as Weight of evidence indicating that also Depanol I has not developmental toxicity potential.

On the one hand, alpha-methylstyrene was tested in SD (Crj:CD) rats in a reproductive/developmental toxicity screening test at doses of 0, 40, 200, and 1000 mg/kg/day by oral gavage administration. The test substance had no effects on reproductive parameters such as the mating index, the fertility index, gestation period, number of corpora lutea or implantations, the implantation index, the gestation index, the delivery index or parturition. Two dams of the 1000 mg/kg/day group lost all their offspring during the lactation period. Parental toxicity was further observed in these females at 1000 mg/kg bw, with target organs being the liver, kidney, and thymus. In the 200 mg/kg/day group, similar changes were observed. On examination of neonates, the 1000 mg/kg/day group showed a decrease of body weight and a slightly low viability index on post-partum day 4 due to the total litter losses of the two dams. There were no significant differences in numbers of offspring or live offspring at birth, the sex ratio, the live birth index, external examination, clinical signs, body weight gain after birth, or necropsy of the offspring. The NOELs for reproductive and developmental toxicity were 1000 mg/kg/day for parental males and 200 mg/kg/day for parental females and offspring.

On the other hand, results from d-limonene in prenatal developmental toxicity studies in rats, mice and rabbits were available in:

- 20 female Wistar rats/group were orally administered 0, 591 and 2869 mg/kg bw/day on days 9–15 of gestation (Adams et al., 2011; Tsuji et al., 1975). Maternal toxicity was noted in the high dose group, including increased mortality (40%) and decreased body weight on gestation day 16 compared to controls; however, by gestation day 20, body weights of high dose dams were not significantly different from controls. No effects were seen in the low-dose group (591 mg/kg bw/day). The offspring of dams in the high-dose group showed several signs of toxicity including significant decreases in body weight in males, absolute and relative weights of the thymus and spleen in males and females, and absolute and relative ovary weights in females. High-dose offspring also exhibited delayed ossification of the metacarpal bone and proximal phalanx. However, any retarded ossification returned to normal within several weeks of birth. In the offspring of low-dose dams, males were reported to have significantly increased relative testes weights, while females exhibited significantly decreased absolute kidney weights compared to controls. Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

- 20 female ICR mice/group were orally administered 0, 591 and 2363 mg/kg bw/day on days 7-12 of gestation. Bodyweights of pregnant mice were recorded during organogenesis (Kodama et al., 1977a; Adams et al., 2011; IRIS 2012). Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. In the remaining 5 mice/group, the number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7. A significant decrease in body weight gain in pregnant mice was observed at 2363 mg/kg. However, no abnormalities were observed in general behavior of the dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg comparing with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of body weight gain was observed in male offspring born to dams dosed orally at 2363 mg/kg, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control. The NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.

- 18 Japanese white rabbits/group were dosed at 0, 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation (Kodama et al., 1977b; Adams et al., 2011; IRIS, 2012). Food consumption and bodyweights of pregnant rabbits were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anomalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of D-Limonene during the gestation. External examination of fetuses showed no anomalies. Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. In conclusion, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be higher than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:
The developmental toxicity study with d-limonene in rats was selected, however other additional weight of evidence was available all showing a consistent results.

Justification for classification or non-classification

Based on the available data, Depanol I does not have to be classified for reproductive and developmental toxicity according the EU labelling regulations Commission Directive 93/21/EEC or CLP regulation No. 1272/2008 of 16 December 2008.