Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well reported study using recognised test methodology with detailed description of findings. Test material was a close chemical analogue of the registered substance

Data source

Reference
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

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Neurodevelopmental toxicity study cohort added to a 2-generation inhalation study investigating toxicity to reproduction. Rats (25/sex/group) were exposed to styrene at 0, 50, 150 and 500 ppm styrene, 6h/day for at least 70 consecutive days prior to mating for F0 and F1 production . Exposure of F0 and F1 females continued through mating and to gestation day 20 . On lactation days 1 through 4, these females were dosed via oral gavage at dose levels designed to mimic peak blood level produced by the inhalation exposures: inhalation exposure then re-commenced, continuing through weaning to postnatal day 21 . Developmental milestones were assessed in F1 and F2 progeny. Neurological development of (randomly selected) F2 pups was investigated using functional observational battery, locomotor activity, acoustic startle response, learning and memory evaluations, together with brain weight and dimension measuremens, and brain morphology/histopathology evaluation.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: vapour
Details on test material:
Styrene (CAS 100-42-5), purity 99.9% minimum, with 10ppm t-butylcatechol inhibitor.

Test animals

Species:
rat
Strain:
other: CrL:CD (SD)IGS BR
Details on test animals and environmental conditions:
Males 263-266g, females 189-190g at start of exposure. Caged (F0, F1 parents individually until pairing) in mesh-bottom cages, but mated females transferred to plastic-bottom cages. Food and water supplied ad lib, except during inhalation exposure.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Whole body vapour exposure, 6h/day for at least 70 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis of test chamber concentrations
Details on mating procedure:
Rats paired 1:1 for up to 14 days; mating confirmed by plug or vaginal smear.
Duration of treatment / exposure:
At least 70 days
Frequency of treatment:
Daily
Duration of test:
F0 males and females: at least 70 consecutive days prior to mating. Exposure of F0 and F1 females continued through mating and to gestation day 20 . On lactation days 1 through 4, these females were dosed via oral gavage at dose levels designed to mimic peak blood level produced by the inhalation exposures: inhalation exposure then re-commenced, continuing through weaning to postnatal day 21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0
Basis:
other: ppm (control). Mean measured 0 ppm (0 mg/l)
Remarks:
Doses / Concentrations:
50
Basis:
other: ppm (nominal). Mean measured concentration 50 ppm (0.21 mg/l)
Remarks:
Doses / Concentrations:
150
Basis:
other: ppm (nominal). Mean measured concentrations F0 151 ppm (0.64 mg/l), F1 153 ppm (0.65 mg/l)
Remarks:
Doses / Concentrations:
500
Basis:
other: ppm (nominal). Mean measured concentrations F0 499 ppm (2.12 mg/l), F1 501 ppm (2.13 mg/l)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Oral dosage given to F0 and F1 females (lactation days 1-4) was calculated to generate peak blood level equivalent to that during 6h inhalation exposure.
Mated females delivered their pups and reared them to weaning. Pup sex was recorded. Where possible 10 (5M, 5F) pups/litter were randomly selected on postnatal day 4: weighing continued to postnatal day 21. Developmental milestones recorded for F1 and F2 pups were: pinna detachment, surface righting response, hair growth, incisor eruption, eye opening, balanopreputial separation, vaginal patency. 1 F2 pup/sex/litter (total 20 pups/sex/group) was assigned to each of 3 groups tested for neurological development.

Examinations

Maternal examinations:
Twice daily cageside observations.
Ovaries and uterine content:
Not included in this developmental assessment.
Fetal examinations:
Not included in this developmental assessment.
Statistics:
Postnatal developmental milestone data were analysed for heterogeneity of variance and normality: parametric analysis of variance was then used to evaluate inter group differences if data were homogeneous and normal (plus Dunnett's test to compare controls with all test groups). If the data were not homogeneous and normal, Kruskal-Wallis nonparametric analysis was applied to evaluate intergroup differences and where appropriate the Mann-Whitney U-test was used to compare the test groups to the controls. Pup weights were analysed by sex, using covariance analysis. Histopathology findings in test and control groups were compared Fisher's Exact test (2-tailed). Functional observation battery data were analysed as for developmental milestone data, except that for descriptive/scalar data Fisher's Exact Test was applied. Other appropriate methods of statistical analysis were applied to locomotor activity assessment and acoustic startle test data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Degeneration of nasal olfactory epithelium at 500 ppm. Reduced bodyweights at 150 and 500 ppm, increased water consumption during gestation (F0, F1) at 500 ppm

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
50 other: ppm mean measured concentration (0.21 mg/l)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
50 other: ppm mean measured concentration (0.21 mg/l)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
Effect level:
150 other: ppm mean measured concentration (0.64 mg/l)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEC
Effect level:
500 other: ppm mean measured concentration (2.13 mg/l)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
Effect level:
500 other: ppm mean measured concentration (2.13 mg/l)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Parental reproductive performance and postnatal survival of pups to weaning were unaffected by styrene exposure.

Up to weaning, F1 pup weights and developmental markers showed no effects of styrene exposure, but subsequent direct exposure at 500 ppm reduced weight gain. Associated with this lower weight gain, delay to balanopreputial separation was observed. Treatment-related effects were more evident in F2 pups than in F1 pups: pre-weaning pup weights were reduced in both 150 and 500 ppm groups and a significant delay in mean age for incisor eruption was seen in the 500 ppm group, together with minor (statistically insignificant) delays to pinna detachment, surface righting, hair appearance, eye opening, and balanopreputial separation. Further evidence of slight developmental delays for F2 animals of the 500 ppm group were also reported: these included apparent changes in locomotor activity development (postnatal days 13-21), reduced swimming ability (postnatal day 24) and reduced grip strength (postnatal days 45 and 60). These F2 effects were considered to represent slight developmental delay resulting from parental styrene exposure at 500 ppm.

Applicant's summary and conclusion

Conclusions:
From this study the authors concluded that repeated inhalation exposure to styrene at up to 500 ppm (2.13 mg/l), a level causing limited maternal toxicity, produced no clear and specific effect on neurological development in rats. No teratogenic action of styrene was seen.
Executive summary:

Exposure of rats to styrene at up to 500 ppm (2.13 mg/l) for at least 70 days produced no evidence of adverse effect on reproductive performance, teratogenicity or embryo-/foeto-toxicity. Some delay to postweaning growth and postweaning developmental milestones (including certain markers of neurological development) was reported, and this was more evident in the F2 generation than in F1 animals. The authors concluded NOAEC values of 500 ppm (2.13 mg/l) for F2 developmental neurotoxicity and 50 ppm (0.21 mg/l) for F2 progeny growth.

Based on the justification document supplied in section 13 of this dossier which supports the use of read-across from styrene to the registered substance 2-phenylpropene, it is concluded that the observed activity profile of styrene and NOAEC values determined in this study can reasonably be considered applicable to the registered substance.