Styrene

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Two Generation Reproduction Study of Styrene by Inhalation in Crl-CD Rats

Author:

Cruzan G. et al.

Year:

2005

Bibliographic source:

Birth Defects Research (Part B) 74: 211-220

Reference 2

Reference Type:

secondary source

Title:

European risk assessment report, Styrene CAS No: 100-42-5 EINECS No 202-851-5, Draft for publication, June 2008 United Kingdom

Author:

European Union

Year:

2008

Bibliographic source:

European risk assessment report, Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Styrene

EC Number:

202-851-5

EC Name:

Styrene

Cas Number:

100-42-5

Molecular formula:

C8H8

IUPAC Name:

ethenylbenzene

Specific details on test material used for the study:

- Name of test material (as cited in study report): styrene
- Physical state: vapour
- Analytical purity: 99.9%
- Impurities (identity and concentrations): benzene, ethylbenzene, styrene oxide, styrene dimers, t-butylcatechol 10ppm (inhibitor of self-reation)

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 Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: (P) x 37 and 38 days
- Weight at study initiation: (P) Males: 263-266 g; Females: 189-190 g; (F1) Males: 97-112 g; Females: 90-104 g
- Fasting period before study: no data
- Housing: clean, wire-mesh cages suspended above cage-board, transferation of the females after mating to plastic maternity cages with nesting material (Bed-O'Cobs CF; The Andersons, Industrial Products Division, Maumee, Ohio)
- Diet: ad libitum
- Water: ad libitum (no water available during inhalation exposure)
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Details on exposure:

No data

Details on mating procedure:

- M/F ratio per cage: no data
- Length of cohabitation: until positive evidence of mating
- Proof of pregnancy: copulatary plug or sperm in vaginal smear
- After successful mating each pregnant female was caged (how): plastic maternity cages with nesting material (Bed-O'Cobs CF; The Andersons, Industrial Products Division, Maumee, Ohio)
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatographic analyses of chamber atmospheres

Duration of treatment / exposure:

70 days (F0 generation); F0 and F1 females continued inhalation exposure throughout mating and gestation through gestation day 20; on lactation days 1 through 4, the F0 and F1 females received styrene via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into 3 equal doses, app. 2 hr. apart)

Frequency of treatment:

6h daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.21, 0.64, 2.13 mg/L (F0); 0, 0.21, 0.64, 2.13 mg/L (F1)
Basis:
analytical conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: oral administration of styrene to F0 and F1 females based on calculation to mimic the peak maternal blood level of styrene during a 6-hr inhalation exposure
- Other:

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, al least 7 hours apart

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

daily

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: yes
[epididymis weight, sperm motility, sperm morphology, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: estrous cycle length, male and female mating index, pre-coital interval, male and female fertility index, gestation length, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
F0 males and females were sacrificed after the mating period and weaning of the F1 pups

GROSS NECROPSY
- Gross necropsy consisted of lung, liver, adrenal, thyroid, spleen and kidney


HISTOPATHOLOGY / ORGAN WEIGHTS
lung, liver, adrenal, thyroid, spleen and kidney

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 28.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs indicated in Table [1] were prepared for microscopic examination and weighed, respectively.

Statistics:

Two-tailed tests for a minimum significance of 5% comparing each test article-treated group to the differences. Parental mating and fertility indices were analyzed using the Chi-square test with Yates' correction factor. Mean parental (weekly, gestation and lactation) and F2 offspring body weight data, food consumption and food ef ficiency data, organ weight data, maternal estrous cycle data, pre-coital intervals, gestation lengths, implantation sites, unacounted sites, ovarian primordial follicle counts, mean number of pups born, live litter size, epididymal and testicular sperm numbers, and sperm production rates were analyzed for heterogeneity of variance and normality. If the data were homogeneous and normal, a parametric one-way analysis of variance (ANOVA) was used to determine intergroup differences. If the results of the ANOVA were significant (p <0.05), Dunnett's test was applied to compare the control group versus all treatment groups. If the data were not homogeneous and normal, the data were analyzed by the Kruskal-Wallis nonparametric ANOVA test to determine the intergroup differences. If the ANOVA revealed statistical significance (p <0.05), the Mann Whitney U-test was used to compare the test article-treated groups to the control group. Pup weights through weaning were analyze separately by sex by a nested analysis of covariance. The number of pups born was used as the covariate.

Reproductive indices:

Male and female mating index
Male and female fertility index

Offspring viability indices:

Live birth index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related findings

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight: decreased mean body weights during premating weeks 3-10, body weight was significantly reduced at week 7 in F0 males


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no indication of adverse effects


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no indication of adverse effects


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) no indication of adverse effects


GROSS PATHOLOGY (PARENTAL ANIMALS): no macroscopic findings attributed to styrene exposure


HISTOPATHOLOGY (PARENTAL ANIMALS): In the 2.13 mg/L males and females, degeneration of the olfactory epithelium that lines the dorsal
septum and dorsal and medial aspects of the nasal turbinates


OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

1

Dose descriptor:

NOAEC

Effect level:

0.64 mg/L air

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

LOAEC

Effect level:

2.13 mg/L air

Sex:

male/female

Basis for effect level:

other: body weight, histopathology (degeneration of olfactory epithelium)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)

CLINICAL SIGNS (OFFSPRING)
No treatment-related findings

BODY WEIGHT (OFFSPRING): (0.64, 2.13 mg/L): reduced after exposure an PND 22-27 in males and females and remained reduced through the F1 exposure period, In the 2.13 mg/L group, F2 birthweights were reduced campared to the control and F2 offspring from both the 0.64- and 2.13 mg/L exposure groups gained weight more slowly than the controls.

SEXUAL MATURATION (OFFSPRING): no indications of adverse effects on reproductive performance

ORGAN WEIGHTS (OFFSPRING): no indications of adverse effects

GROSS PATHOLOGY (OFFSPRING): no macroscopic findings attributed to styrene exposure

HISTOPATHOLOGY (OFFSPRING): In the 2.13 mg/L males and females (F1), degeneration of the olfactory epithelium that lines the dorsal septum and dorsal and medial aspects of the nasal turbinate

OTHER FINDINGS (OFFSPRING):

Effect levels (F1)

1

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.64 mg/L air

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

2.13 mg/L air

Sex:

male/female

Basis for effect level:

other: body weight; histopathology (degeneration of olfactory epithelium)

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEC

Generation:

F2

Effect level:

0.21 mg/L air

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Dose descriptor:

LOAEC

Generation:

F2

Effect level:

0.64 mg/L air

Sex:

male/female

Basis for effect level:

other: body weight

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The principal finding of the study in the parental animals of the F0 and F1 generations was the degeneration of the olfactory epithelium lining of the nasal cavity observed in the high-exposure group only. The degeneration was characterized by disorganisation and one or more of the following features: regenerative hyperplasia, individual cell necrosis and atrophy. There was also an increased presence of Bowman’s glandular elements and cysts in the olfactory epithelium. The incidence and degree of degeneration were less pronounced in the F1 generation compared to the F0 generation. No evidence of this lesion was found at the lower concentration levels in either the F0 or F1 generations. In the high-exposure group of both the F0 and F1 generations, the mean body weight of the males was statistically significantly reduced by 7-8% (F0) and 8-13% (F1) compared to controls and in females by 7-8%. Mean body weights of the high-exposure females during gestation were reduced by 5% (not statistically significant) in the F0 generation and by 6 -7% (statistically significant) in the F1 generation, indicating that there were statistically significant maternal effects on body weight only in the high-exposure females of the F1 generation. In the mid-exposure group of the the F0 and F1 generations the mean body weight of the males was statistically significantly reduced by 6-7%. There were no statistically significant effects on body weights in the mid-exposure females of the F0 and F1 generations, although reductions of up to 5-6% were observed in the F1 females during the study including gestation. Overall, there were no statistically significant maternal effects on body weight at 0.64mg/L. Water consumption measured in females during the gestation and lactation periods only was statistically significantly increased during gestation in the midand high-exposure groups of the F1 generation (by 11-14% and 20-24% respectively) and in the high-exposure group of the F0 generation (by 13-24%). Relative (to final body weight) liver weights were statistically significantly increased compared to controls in the 0.64 and 2.13mg/L F0 males by 6% and 15% respectively and in the 2.13 mg/L F1 males by 11.6% but the absolute liver weights were similar to controls. Also, statistically significant increases in relative weights of the brain were observed in the high-exposure males of the F0 generation (by 7.4% of the control value) and in the mid- and high-exposure males of the F1 generation (by 9.6% and 6.4% respectively compared to controls), but again the absolute weights were similar to controls. The relative weight of the kidneys was statistically significantly increased in the mid- and high-exposure males of the F0 generation by 5.8% and 10% respectively, but the absolute weights were similar to controls. All these increases in relative organ weights can be attributed to the decrease in mean body weights observed in these groups of animals, and hence, are considered of no toxicological significance. Reproductive performance (i.e. mating behaviour and fertility), gestation length, litter data (number of pups, sex ratio), postnatal survival, sperm evaluations and primordial follicle counts were not adversely affected by styrene exposure across the generations. The mean length of the estrous cycle in the high-exposure females of the F0 generation was shorter (4.2 days) and differed statistically from that of controls (5.8 days). However, the value was similar to the laboratory’s historical control mean value (4.3 days) and within the historical control range (4.1-5.1 days) and not affected in subsequent generations. Hence, it is not considered to be exposure-related. No exposure-related effects were observed on the pre-weaning body weights of the F1 pups (PND 1-21). However, the body weights of the high-exposure F1 pups were decreased compared to controls (by 7-7.6%, not statistically significant) during the post-weaning period (PND22-28) and the bodyweight gain in this group was statistically significantly lower than

that of the controls (by 11%). A delay (approx. 2 days) in preputial separation was observed in the high-exposure F1 males. The correlation between body weight and preputial separation in rats is clearly established; therefore, the delay in preputial separation observed in the high-exposure group of the F1 generation is likely to be a consequence of the decrease in body weight observed in this group following direct exposure to styrene after weaning (PND22-28). The mean absolute testes weights were slightly lower in all F1 males exposure groups by approximately 5-7%, but the observation did not follow a dose-response relationship. The differences from the controls were statistically significant for the left and right testis in the high-exposure group and for the left testis in the low-exposure group. In the absence of any associated morphological (macroscopic and microscopic) alteration and considering the lack of a dose-response relationship, the small magnitude of the decrease and the absence of such an effect in the F0 and F2 males, this finding is considered to be of no toxicological significance. The general physical condition, sex ratios and survival to weaning of the F2 generation were unaffected by maternal exposure to styrene at all levels tested. Statistically significant, exposure-related decreases in body weight of 10-13% and 7-10% were observed in the high- and mid- exposure pups of the F2 generation respectively throughout the pre-weaning period (PND 0-21). The reductions in body weight of the F2 pups in the high-exposure group continued throughout the post-weaning period even though exposure had stopped. No macroscopic findings attributable to exposure were evident at necropsy. Statistically significant reductions in mean absolute pituitary gland weight compared to controls were noted in the high-exposure F2 male pups (by 34%) and in the mid- and high-exposure F2 female pups (by 19% and 24% respectively). Also, the mean relative (to final body weight) pituitary gland weight was statistically significantly reduced in the high-exposure F2 male pups by 22%. Other effects included statistically significant decreases in the mean absolute thymus and uterine weights by 18% of the control values in the high-exposure F2 female pups and a statistically significant increase in the mean relative brain weight by 11% of the

control value in the same group. Although the magnitude of the decreases observed in the pituitary gland weight at high- and mid-exposure levels and in the thymus and uterine weight at the high exposure is relatively large and cannot be completely accounted for by the reduced body weights observed in these pups, in the absence of information on the normal growth rate of these organs in fast-developing organisms and on its relationship to body weight development, their toxicological significance cannot be determined. However, given the lack of any associated histopathology, it is reasonable to assume that these pup organ weight reductions are unlikely to represent adverse developmental effects of styrene exposure. The attainment of the pre-weaning developmental landmarks (pinnal detachment, surface righting response, incisor eruption and hair growth) and the acquisition of the preputial separation were also slightly delayed in the high-exposure F2 pups. It is considered that these effects were probably due to the slight delay in growth (reduced body weights) observed in these pups.