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

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-phenylpropene

EC Number:

202-705-0

EC Name:

2-phenylpropene

Cas Number:

98-83-9

IUPAC Name:

isopropenylbenzene

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 1-Methylethenylbenzene; alpha-Methylstyrene; isopropenylbenzene
- Molecular formula (if other than submission substance): C9-H10
- Molecular weight (if other than submission substance): 118.18 g/mol
- Smiles notation (if other than submission substance): c1(ccccc1)C(C)=C
- InChl (if other than submission substance): 1/C9H10/c1-8(2)9-6-4-3-5-7-9/h3-7H,1H2,2H3
- Structural formula attached as image file (if other than submission substance): See illustration attached
- Substance type: Organic monoconstituent
- Physical state: Liquid
- Analytical purity: 99.6%
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: Not applicable (monoconstituent)

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

3

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.