Styrene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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/report meeting basic scientific principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Developmental neurotoxicity study of styrene by inhalation in Crl-CD rats.

Author:

Cruzan G. et al.

Year:

2005

Bibliographic source:

Birth Defects Research (Part B) 74: 221-232

Reference 2

Reference Type:

secondary source

Title:

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

Author:

European Union

Year:

2007

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

Principles of method if other than guideline:

This study was part of the main reproductive toxicity study and conducted to assess potential adverse functional and/or morphological effects of styrene on the neurological system in the F2 offspring following F0 and F1 generation whole-body inhalation exposures. Four groups of male and female rats (25/sex/group) were exposed to 0, 0.21, 0.64, and 2.13 mg/L styrene for 6 hr daily for at least 70 consecutive days prior to mating for the F0 and F1 generations . Inhalation exposure continued for the F0 and F1 females throughout mating and through gestation day 20 . On lactation days 1 through 4, the F0 and F1 females received styrene in virgin olive oil via oral gavage at dose levels of 66, 117, and 300 mg/kg/day (divided into three equal doses, approximately 2 hr apart). Inhalation exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through weaning of the F1 or F2 pups on postnatal day (PND) 21 . Developmental landmarks were assessed in F1 and F2 offspring . The neurological development of randomly selected pups from the F2 generation was assessed by functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, brain weights and dimension measurements, and brain morphometric and histologic evaluation.

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
- Analytical purity: 99.9%
- Impurities (identity and concentrations): benzene, ethylbenzene, styrene oxide, styrene dimers, t-butylcatechol 10 ppm (inhibitor of self-reation)

Test animals

Species:

rat

Strain:

Crj: CD(SD)

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: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Details on exposure:

No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatographic analyses of chamber atmospheres.

Details on mating procedure:

- M/F ratio per cage: no data
- Length of cohabitation: until positive evidence of mating
- Proof of pregnancy: capulatary 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:

Duration of treatment / exposure:

F0: 70 d premating, mating, 20 d gestation, 21 d until weaning of F1 pups (PND 1-4 oral gavage in virgin olive oil, three equal doses approx. 2 hrs apart)
F1: 70 d premating, mating, 20 d gestation, 21 d until weaning of F2 pups (PND 1-4 oral gavage in virgin olive oil, three equal doses approx. 2 hrs apart)

Frequency of treatment:

6 hr daily

Duration of test:

252 days

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

Examinations

Statistics:

F2 mean day of acquisition of preweaning/post-weaning developmental landmarks data 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 sta tistical 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 analyzed separately by sex by analysis of covariance (ANCOVA), with pups weights nested within the litter, with the litter size the covariate. The number of pups born was used the covariate. Histopathologic findings in the test article-treated groups were compared to the control group using a two-tailed Fisher's Exact test. FOB data were subjected to a parametric one-way analysis of va riance (ANOVA) to determine intergroup differences . If statistically significant differences were indicated by the ANOVA, Dunnett's test was used to compare the control and treated groups. FOB parameters which yielded scalar and descriptive data, were analyzed by Fisher's Exact Test. Intrasesion total counts measured in thelocomotor activity assessment and intrasession peak response and latency to peak response measured in the acoustic startle assessment were analyzed by the univariate repeated measures ANOVA to determine the presence of an interaction effect treatment group by time using a Geisser-Greenhouse adjusted F-statistic. If a significant interaction effect of treatment group by time was indicated by the ReMA NOVA, Dunnett's test was used to compare the control and treated groups at each within-session interval.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

>= 2.13 mg/L air

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

0.21 mg/L air

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

LOAEC

Effect level:

0.64 mg/L air

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 2.13 mg/L air

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

There were exposure-related reductions in mean body weights of the F1 and F2 offspring from the mid and high-exposure groups and an overall pattern of slightly delayed development evident in the F2 offspring only from the 2.13 mg/L group. This developmental delay included reduced body weight (which continued through day 70) and slightly delayed acquisition of some physical landmarks of development. Styrene exposure of the F0 and F1 animals had no effect on survival, the clinical condition or necropsy findings of the F2 animals. No direct effects of styrene exposure on absolute brain weights of PND 21 or 72 male and female rats were noted. Brain weights relative to final body weights of 2.13 mg/L group females were increased compared to control females in these same animals because mean final body weights were slightly decreased in the 2.13 mg/L group females. Slight, but statistically significant decreases (by 4% of the control value) in mean brain length occurred on PND 21 in females from the mid- and highexposure groups but the brain width and absolute brain weight were similar to control group values. No microscopic findings that could be attributed to parental exposure to styrene were noted in the 2.13 mg/L group as the result of the qualitative neuropathologic examination of the brain on PND 21 or central and peripheral nervous system tissues on PND 72. There were no histomorphologic changes in measurements of brain regions on PND 21 or 72 that could be attributed to parental exposure in the 2.13 mg/L group. In female rats of the 2.13 mg/L group evaluated on PND 21, the mean height of the hemisphere on Level 1 was slightly (6%) increased . However, the cortical thickness was not altered when compared to the control group, and the height of the hemisphere was not altered in offspring evaluated on PND 72. Functional observational battery evaluations conducted for all Fl dams during the gestation and lactation periods and for the F2 offspring were unaffected by styrene exposure. Swimming ability as determined by straight channel escape times measured on PND 24 were increased, and reduced grip strength values were evident for both sexes on PND 45 and 60 in the 2.13 mg/L group compared to controls. There were no other parental exposure-related findings in the F2 pre-weaning and post-weaning functional observational battery assessments, the PND 20 and PND 60 auditory startle habituation parameters, in endpoints of learning and memory performance (escape times and errors) in the Biel water maze task at either testing age, or in activity levels measured on PND 61 in the 2.13 mg/L group. Taken together, the exposure-related developmental and neuromotor changes identified in F2 pups from dams exposed to 2.13 mg/L occurred in endpoints known to be both age- and weight-sensitive parameters, and were observed in the absence of any other remarkable indicators of neurobehavioral toxicity.