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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2003 to 12 December 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is a single generation investigation rather than a two generation study as specified in REACH test guidelines. However, in the absence of any adverse findings and the EU indications of moving to replace the 2-generation study with data from a single generation study, it is considered that the study presented here adequately addresses the requirements of this annex point.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
The objective of this study was to evaluate the effects of the test substance on the reproductive function, mating behaviour, maturation of gametes, fertilization, and embryonic development when administered to male and female Crl:CD®(SD)IGS BR rats via inhalation during premating, mating, and conception to implantation. This study also evaluated sperm parameters (count, motility, and morphology), and histopathology in reproductive tract and other potential target organs. The inhalation route is the route of potential human exposure.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,3-dichloro-1,3-butadiene (DCBD); synonyms 2,3-dichlorobutane; dichlorobutadiene
- CAS number 1653-19-6
- Substance type:
- Physical state: clear to straw-yellow liquid
- Analytical purity: 98.2% (H-26033) and 98.6% (H-26034)
- Impurities (identity and concentrations): H-26033 2,4,4-trimethyl-1-pentene or DIB-1 0.83%
H-26033 2,4,4-trimethyl-2-pentene or DIB-2 0.20%
H-26034 2,4,4-trimethyl-1-pentene or DIB-1 0.70%
H-26034 2,4,4-trimethyl-2-pentene or DIB-2 0.17%

- Lot/batch No.: H-26033 and H-26034
- Expiration date of the lot/batch: not specified but materials were used rapidly to avoid polymerisation issues and so expiration of the batch was not relevant to the conduct of the study

- Stability under test conditions: The test substance is inherently reactive and may spontaneously polymerize (unless inhibited) or dimerize. Storage conditions were chosen to minimize sample degradation. Based on purity analyses performed prior to and immediately after study conduct, no evidence for instability of the test substance was detected. Analysis of the liquid test substance however showed the concentrations of mixed dichlorobutadiene-based dimers had increased with time but were less than 1,000 ppm, the maximal level considered acceptable for this study.
- Storage condition of test material:
The test substance, DCBD, is a reactive liquid and may spontaneously polymerize (unless inhibited) or dimerize. To minimize sample degradation, polymerization inhibitors were added to the test substance prior to shipping, and the test substance was shipped on ice. Upon receipt, the test substance was stored under nitrogen in a freezer maintained at approximately -20°C except when used to generate test atmospheres and when being analyzed.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:CD (SD) IGS BR rats obtained from Charles River Laboratories Inc, Raleigh, North Carolina. 115 male and 115 female rats obtained on 4 September 2003 (birth date was 23 July 2003) aged six weeks on arrival. Weight range on arrival was 142.7 to 189.9g (males) 127.2 to 161.8g (females)

- Age at study initiation: (P) 54 days -
- Weight at study initiation: 229.9 to 292.6g males and 163.5 to 213.5g females

- Fasting period before study: No
- Housing: pre-test and pre-mating rats were housed individually in suspended stainless steel wire-mesh cages. During cohabitation rats were housed in pairs in suspended wire-mesh cages. After mating the females were individually housed in polycarbonate pans if no evidence of copulation found, pregnant dams were housed in wire-mesh cages with cageboards throughout gestation and from GD 20 to delivery/sacrifice the females were housed in polycarbonate pans with bedding

- Diet (e.g. ad libitum): pelleted PMI Nutrition International Certified Rodent Diet 5002 ad libitum
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70% RH
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12/12 1600-1800 h


IN-LIFE DATES: From: 15 September 2003 To: 12 December 2003

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: test substance saturated nitrogen gas mixed with filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chambers were constructed of stainless steel and glass, were cubical with square pyramidal chamber inlets and outlets (NYU style), with a tangential feed at the chamber inlet to promote vapour mixing and uniform distribution of the test substance atmosphere. The internal nominal volume of each chamber was 1.4 m³. Homogeneous distribution of DCBD vapour for this type of exposure chamber was verified
- Method of holding animals in test chamber: rats held individually in wire mesh stainless steel cages allowing whole body exposure, except during cohabitation when pairs were housed in same cages
- Source and rate of air:Filtered room air

- System of generating vapour: the test material was held in a glass container in a low temperature (circa -20 to -7°C) water bath to control evaporation rate and polymerisation or dimerisation
- Temperature, humidity, pressure in air chamber:
- Air flow rate: Nitrogen flows ranged from 0.08 - 0.23, 0.15 - 0.37, or 1.3 - 2.8 L/min for the 1, 5,
or 50 ppm chambers, respectively
- Air change rate: 10 air changes per hour

- Treatment of exhaust air: discharged directly to exhaust stack


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography at hourly intervals throughout exposure periods. Known volumes of chamber atmosphere drawn directly from animals breathing zone injected directly into gas chromatograph equipped with a flame ionisation detector
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: one to one
- Length of cohabitation: two weeks
- Proof of pregnancy: Not specified
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in stainless steel mesh cage with cageboard during gestation and transferred to polycarbonate pans with bedding on GD 20 to termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance, DCBD, is a reactive liquid and may spontaneously polymerize (unless inhibited) or dimerize. To minimize sample degradation, polymerization inhibitors were added to the test substance. Because of the duration of this study and the reactive nature of the test substance, it was necessary to use 2 separate production lots of DCBD. After every 5 exposures, any DCBD remaining in the generation flask was discarded and replaced with fresh DCBD. The amounts of DCBD added to the generation flask were sufficient to generate test atmospheres for the greatest number of days while ensuring that the amount of spontaneously formed DCBD-dimers remaining in the flask was within acceptable levels (< 1000 ppm).
Purity/stability analyses and dimer quantification of H-26033 and H-26034 were performed by capillary gas chromatography. Mass spectral identification was performed by Gas Chromatography/Mass Spectroscopy (GC/MS) for all peaks > 0.1% normalized area percent.
Purity and stability analyses were completed by GC and dimer quantification was similarly completed using GC for samples of both batches of test material. Mass spectral identification was completed using GC/MS for the undiluted test material.
Chamber atmosphere concentration and characterisation were completed using hourly samples collected from the animals breathing zone in the exposure chambers. Analysis was by GC.


Duration of treatment / exposure:
Premating (Males and Females) 6 hours/day, 5 days/week over 56 days = 40 Exposures
Cohabitation (Males and Females) 6 hours/day, 7 days/week for up to 14 days
Post Cohabitation (Males and Nonpregnant Females) 6 hours/day, 7 days/week for approximately 7 days
Gestation (Pregnant Females) 6 hours/day, 7 days/week from gestation Day 0 – 7
Frequency of treatment:
6 hours per day, exposed on daily basis
Details on study schedule:
Test days 1-56 = premating treatment phase
Test days 56-70 = cohabitation phase
Test days up to day 77 = treatment of pregnant females

Bodyweights recorded on day after arrival and twice during acclimatisation, at weekly intervals during pre-mating, cohabitation and for the males and non-pregnant females post cohabitation. Pregnant females were weighed during gestation on days 1, 7, 14 and 21.

Physical examinations were conducted at same times as bodyweights recorded.

Food consumption was not recorded during the pre-test, but at weekly intervals in pre-mating treatment period and at the same intervals as bodyweight recording during gestation.

Oestrous cycling evaluations - vaginal lavage samples collected daily from all females from 3 weeks prior to the beginning of cohabitation until copulation was confirmed or cohabitation ended.

Sperm parameters were evaluated in all males surviving to scheduled termination
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 5 and 50 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
24 males and 24 females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
Dose selection was based on the results from several previously conducted studies in which reproductive and developmental toxicity was investigated at dose concentration up to circa 50 ppm. 50 ppm was selected as the high concentration for this study since maternal and foetal toxicity were expected to be observed. The intermediate exposure concentration was 5 ppm, and the low exposure concentration was 1 ppm, since it was considered unlikely that maternal or foetal toxicity would occur at these concentrations.

- Rationale for animal assignment (if not random): Rats of each sex were ranked by their most recently recorded body weight and randomly assigned to control and experimental groups. The randomization resulted in a distribution in which the mean body weights for all groups within a sex were not statistically different.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout all phases of treatment


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: checked daily


BODY WEIGHT: Yes
- Time schedule for examinations: day after arrival and a further twice during acclimation. at weekly intervals from beginning of pre-mating treatment. On days 0, 7, 14 and 21 of gestation


FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Feeders weighed at beginning and end of each week. Consumption and feed efficiency calculated.
Oestrous cyclicity (parental animals):
Vaginal lavage samples were collected daily from all female rats in order to determine the stages of the oestrous cycle. Vaginal lavage samples were collected beginning 3 weeks prior to start of cohabitation and continuing until copulation was confirmed or the cohabitation period ended.
The vaginal lavage sample collected on the day copulation was confirmed was not used for estrous cycle evaluation.
Vaginal lavage samples were examined microscopically for determination of the stage of oestrous cycle (dioestrous, oestrous, or proestrous).
Sperm parameters (parental animals):
Parameters examined in all parental males surviving to scheduled termination right epididymis weight; percentage of motile sperm per 200 cells per animal; sperm smears produced to analyse sperm morphology - abnormal head, tail - and percent normal sperm; sperm count per cauda epididymis and per gram cauda epididymis; spermatid count per gram testis and per testis were determined.
Litter observations:
PARAMETERS EXAMINED
Intact and empty uterus of each female with at least one viable foetus was weighed and bodyweight adjusted for gravid uterus weight. The corpora lutea count was recorded. The types of implantation - live or dead foetuses and resorptions recorded

The following parameters were examined in F1 offspring:
[number and sex, weight and external alterations of pups


GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving parental animals terminated after cohabitation phase
- Maternal animals: All surviving animals terminated on gestation day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Organ weights recorded for lungs, liver, brain, kidneys, spleen, thymus and pituitary gland from males and females. In addition, testes, epididymides, seminal vesicles and prostate were weighed for males and intact and empty uterus and ovary weights recorded for females.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination. Tissues were examined for the first ten males and ten females of the control and high dose groups. Only organs showing treatment related effects were examined for the intermediate and low dose groups - in this study this was limited to examination of nasal tissues:


The following tissues and all gross lesions were collected from all animals sacrificed by design, and preserved in appropriate fixative for possible future histopathology examination.

Digestive System Cardiovascular System Musculoskeletal System
Liver heart skeletal muscle
oesophagus aorta femur/knee joint
stomach sternum
duodenum Hematopoietic System
jejunum spleen Reproductive System
ileum thymus Male
caecum mandibular lymph node Testes
colon mesenteric lymph node epididymides
rectum bone marrow prostate
salivary glands seminal vesicles
pancreas coagulating glands
pituitary gland
Endocrine System
Urinary System thyroid gland Female
kidneys parathyroid glands ovaries(with oviducts)
urinary bladder adrenal glands uterus
vagina
Respiratory System Nervous System cervix
lungs brain (including cerebrum,
trachea cerebellum, medulla/pons) Miscellaneous
nose spinal cord (cervical, skin
larynx mid-thoracic, lumbar) eyes (including retina and optic nerve)
pharynx sciatic nerve gross observationse






Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to post mortem 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 tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Descriptive statistics were analysed for the various parameters investigated in the study.
Levene's test for homogeneity and Shapiro-Wilk test for normality
One way analysis of variance followed by Dunnetts test
Kruskal-Wallis test followed by Dunn's test
Cockridge-Armitage trend test as appropriate
Reproductive indices:
The indices of reproductive function that were calculated:
number of corpora lutea - number of implantation sites
preimplantation Loss (%)a = number of corpora lutea x 100
number of implantation sites - number of fetuses
postimplantation Loss (%)a = number of implantation sites x 100
a - Determined for each litter. Mean and standard deviation for each dose level were calculated

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

Oestrous cycle parameters (percent days in dioestruos, proestruos, and oestruos) and oestrous cycle length were evaluated prior to and during the cohabitation period (for at least 3 weeks) in P1 rats. Necropsy of the P1 females was conducted on GD 21 and the following parameters evaluated:
-gross pathological examination, number and status of implantation sites, and foetal assessments (viability, location, sex, foetal weight, external alterations). All P1 male rats were given a gross pathological examination at the end of the cohabitation period. Epididymal sperm motility, morphology and counts, and testicular spermatid counts were determined in all P1 male rats. Reproductive organs and other selected organs, including potential target organs, were weighed and collected for all P1 rats. Potential target organs (liver, spleen, thymus, kidneys, urinary bladder, lungs, trachea, nose, larynx, pharynx, pituitary gland) and gross lesions from P1 rats in the control and 50 ppm groups (10/sex/group) were examined microscopically; tissues in the low and intermediate groups were subsequently evaluated as needed to determine a no-adverse-effect level.

The parameters assessed but showing no effects of treatment or no adverse effects of exposure to DCBD included:
mortality and clinical observations in P1 males and females
oestrous cycle and sperm parameters in P1 rats
mating, precoital interval and fertility in P1 rats
number of corpora lutea; implantation sites, resorptions, live foetuses; pre- and postimplantation loss
foetal weight, sex ratio, and external alterations
gross observations in P1 male and females
organ weights in P1 females

Effects considered to be related to DCBD exposure were evident in the high concentration group - 50 ppm:
decreased body weight, weight gain, food consumption, and food efficiency in P1 males and females
degeneration of the nasal olfactory epithelium in P1 rats

Under the conditions of this study, the no-observed-effect level (NOEL) for reproductive and developmental toxicity was 50 ppm, the highest concentration tested. The NOEL for effects in P1 rats was 5 ppm based on adverse effects on body weight and food consumption parameters, and nasal olfactory epithelial toxicity at 50 ppm.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEC
Effect level:
>= 5 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: based on adverse effects on body weight and food consumption parameters, and nasal olfactory epithelial toxicity at 50 ppm.
Dose descriptor:
NOAEC
Effect level:
>= 50 ppm (analytical)
Sex:
male/female
Remarks on result:
other: Generation: reproductive and developmental effects (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

Foetal weight, sex ratio, and external alterations showed no treatment related changes in this study. The high dose of 50 ppm had no adverse effects on the offspring.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Effect Level for inhalation exposure in the parental generation was 5 ppm based on adverse effects on bodyweight and food consumption and indications of nasal olfactory epithelium toxicity at the high dose, 50 ppm.
For reproduction and developmental effects the NOEL was 50 ppm , the highest dose tested.
Executive summary:

A single generation fertility and early embryonic developmental study was conducted with 2,3-dichloro-1,3- butadiene (DCBD). Groups of Crl:CD®(SD)IGS BR rats (24/sex/concentration) were exposed by inhalation (whole body) at mean vapour concentrations of 0, 1, 5, or 50 ppm. Rats were exposed for 6 hours/day during premating (8 weeks; 5 days/week), during cohabitation of mating pairs (up to 2 weeks, 7 days/week), and then post-cohabitation for males and nonpregnant females (approximately 7 days, 7 days/week). The pregnant dams were exposed from conception to implantation (days 0-7 of gestation).

Clinical observations, body weight, and food consumption were determined at weekly intervals through premating and gestation. Following at least 50 days of exposure (premating), males and females (P1 parental generation) were cohoused within their respective treatment groups until evidence of copulation was observed or 2 weeks had elapsed with no evidence of mating.

Oestrous cycle parameters (percent days in dioestruos, proestruos, and oestruos) and oestrous cycle length were evaluated prior to and during the cohabitation period (for at least 3 weeks) in P1 rats. Necropsy of the P1 females was conducted on GD 21 and the following parameters evaluated:

-gross pathological examination, number and status of implantation sites, and foetal assessments (viability, location, sex, foetal weight, external alterations). All P1 male rats were given a gross pathological examination at the end of the cohabitation period. Epididymal sperm motility, morphology and counts, and testicular spermatid counts were determined in all P1 male rats. Reproductive organs and other selected organs, including potential target organs, were weighed and collected for all P1 rats. Potential target organs (liver, spleen, thymus, kidneys, urinary bladder, lungs, trachea, nose, larynx, pharynx, pituitary gland) and gross lesions from P1 rats in the control and 50 ppm groups (10/sex/group) were examined microscopically; tissues in the low and intermediate groups were subsequently evaluated as needed to determine a no-adverse-effect level.

The parameters assessed but showing no effects of treatment or no adverse effects of exposure to DCBD included:

mortality and clinical observations in P1 males and females

oestrous cycle and sperm parameters in P1 rats

mating, precoital interval and fertility in P1 rats

number of corpora lutea; implantation sites, resorptions, live foetuses; pre- and postimplantation loss

foetal weight, sex ratio, and external alterations

gross observations in P1 male and females

organ weights in P1 females

Effects considered to be related to DCBD exposure were evident in the high concentration group - 50 ppm:

decreased body weight, weight gain, food consumption, and food efficiency in P1 males and females

degeneration of the nasal olfactory epithelium in P1 rats

Under the conditions of this study, the no-observed-effect level (NOEL) for reproductive and developmental toxicity was 50 ppm, the highest concentration tested. The NOEL for effects in P1 rats was 5 ppm based on adverse effects on body weight and food consumption parameters, and nasal olfactory epithelial toxicity at 50 ppm.