Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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:
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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
Although not stated in the published paper, test method was closely similar to OECD 416
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 (contained 10 ppm t-butylcatechol inhibitor)

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
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 femaled 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
Details on mating procedure:
Sexes paired 1:1 for up to 14 days; mating confirmed by plug or vaginal smear
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis of chamber atmospheres
Duration of treatment / exposure:
F0 parents: 70 days; F0 and F1 females: exposure continued through mating and gestation to gestation day 20 but on lactation days 1-4 these females were dosed via oral gavage (66, 117, or 300 mg/kg/day, split into 3 equal doses ca. 2h apart). F1 pups: exposure started on postnatal day 22.
Frequency of treatment:
Daily (6h/day)
Details on study schedule:
Continuous exposure as detailed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal conc.
Mean measured 0 ppm
Remarks:
Doses / Concentrations:
50 ppm
Basis:
nominal conc.
mean measured F0 and F1 50 ppm/0.21 mg/l
Remarks:
Doses / Concentrations:
150 ppm
Basis:
nominal conc.
mean measured F0 151 ppm (0.64 mg/l), F1 153 ppm (0.65 mg/l)
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal conc.
mean measured 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. Litters standardised (random selection to give 5M, 5F where possible) on postnatal day 4.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
Twice daily observations of parents plus mid-point of exposure check in situ. Bodyweights and food consumption recorded weekly (females food and water consumption more frequently during gestation and lactation).
Oestrous cyclicity (parental animals):
Monitored by vaginal smears
Sperm parameters (parental animals):
At necropsy: right epididymides plus left testes with cauda epdidymides weighed, sperm morphology and motility assessed. Homogenisation-resistant sperm count and sperm production rate also determined
Litter observations:
Postnatal deaths necropsied up to weaning and postnatal days 22-28. Pups sexed and developmental milestones recorded.
Livebirth numbers and postnatal deaths were recorded, together with pup weights.
Postmortem examinations (parental animals):
Ovarian sections collected from all F0 females; 5 ovarian sections from control and high-dose F1 females subjected to histopathological examination. Following complete necropsy investigations including organ weight recording, selected tissues taken for histopathology: microscopic examination applied to tissues from control and high-dose F0, F1 parents plus decedents from intermediate test groups
Postmortem examinations (offspring):
Gross abnormalities (internal or external) recorded at necropsy, with organ weights
Statistics:
Two-tailed tests compared each test group to control. Parental mating and fertility indices analysed by Chi-squared test with Yates' correction. Other parental and F2 progeny data were analysed for heterogeneity of variance and normality. If homogeneous and normal, parametric one-way analysis of variance was used to determine intergroup differences: then either Dunnett's test or the Kruskal-Wallis nonparametric test was applied to determine intergroup differences. TheMann Whitney U-test was used to compare test groups to the control where appropriate. Pup weights during weaning were analysed separately (nested analysis of covariance, by sex).
Reproductive indices:
Parameters examined: oestrous cycle length, male and female mating index, pre-coital interval, male and female fertility index, gestation length, pup number and sex, stillbirths, livebirths, postnatal mortalities, presence of gross anomalies, bodyweight gain, physical or behavioural abnormalities.
Reproductive indices: mating, fertility
Offspring viability indices:
Livebirths, postnatal mortality

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced at 500 ppm in F0 males and females (premating weeks 3-10); reduced at 150 ppm in F0 males (gain during week 1 and in week 7) and F1 males/females ( postnatal days 22-27 and through F1 exposure).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slightly reduced at 500 ppm in F0 males and females (premating weeks 3-10); reduced at 150 ppm in F0 males (gain during week 1 and in week 7) and F1 males/females ( postnatal days 22-27 and through F1 exposure).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight-relative liver weights increased at 150 and 500 ppm (F0, F1 males). No other effects
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nasal olfactory epithelium degeneration at 500 ppm

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Limited evidence of systemic toxicity at 150 ppm; no effects on reproductive parameters

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
50 other: ppm mean measured concentration (0.21 mg/l)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity: degeneration of nasal olfactory epithelium at 500 ppm. Reduced bodyweights (F0+F1 males, F1 females at 150 and 500 ppm), increased water consumption during lactation (F0 females, 150 and 500 ppm)
Dose descriptor:
NOAEC
Effect level:
500 other: ppm mean measured concentration (2.1 mg/l)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity: no effects on fertility, reproductive organs or sperm, gestation, live litter size or postnatal survival seen at highest tested concentration
Remarks on result:
other: Generation: F0, F1 (migrated information)

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):
Mean F2 male/female birthweights and bodyweight gains during pre-weaning reduced
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F2 male bodyweight-relative pituitary weights reduced at 500 ppm
Gross pathological findings:
no effects observed

Details on results (F1)

F2 birthweights and pre-weaning weight gains reduced in high-dose group only. Possible F2 male pituitary weight reduction effect observed. No other effects recorded.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Although significant reductions in pituitary weight were seen at 500 ppm in F2 male pups and at 150 and 500 ppm in F2 females, only in F2 males at 500 ppm where bodyweight-relative pituitary weight reduction was also seen was a pituitary effect considered attributable to styrene exposure. The other cases, together with statistically significant decreases in mean thymus and uterine weights in F2 females at 500 ppm, were judged likely to be associated with growth retardation.

Applicant's summary and conclusion

Conclusions:
No significant effect on reproductive performance, or on morphology or function of reproductive organs, was seen when rats were exposed to styrene (6h/day over 2 generations) at concentrations up to 500 ppm (mean measured concentration: equal to 2.1 mg/l) in this study. A NOAEC value of 50 ppm/0.21 mg/l was concluded for systemic toxicity to the F0 and F1 parental generations , togther with a NOAEC value of 2.1 mg/l for toxicity to reproduction.
Executive summary:

In this reliable study of styrene toxicity, no significant effect on reproductive performance, or on morphology or function of reproductive organs, was seen when rats were exposed to styrene (6h/dayover 2 generations) at concentrations up to 500 ppm (mean measured concentration: equal to 2.1 mg/l) in this study. A NOAEC value of 50 ppm/0.21 mg/l was concluded for systemic toxicity to the F0 and F1 parental generations, together with a NOAEC value of 2.1 mg/l for toxicity to reproduction. 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 these NOAEC values and the observed absence of toxicity to reproduction can reasonably be considered applicable to the registered substance.