Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints.)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
yes
Remarks:
(Study included FOB and motor activity endpoints.)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No
- Housing:
During Non-Exposure Periods: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation, gestation/lactation as described below.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
Gestation/Lactation: Females presumed pregnant were housed individually in solid bottom caging with bedding and enrichment. During lactation periods, adult females were housed with their litters. Enrichment was not provided during the approximate period of expected delivery.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: filtered and conditioned air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style) with a nominal internal volume of 350 L
- Method of holding animals in test chamber: individually placed in stainless steel wire-mesh modules (one/module) and exposed, whole-body, inside the exposure chamber; during the mating period animals were housed as mating pairs and exposed in the same manner.
- Source and rate of air: filtered and conditioned air and oxygen
- System of generating vapour:test substance vapour and supplemental oxygen were metered into a 1L 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 19–24°C; 41–82%; pressure not reported
- Air flow rate: 70–114 L/min
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: to fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by gas chromatography at approximately 30-minute intervals in the test chambers. A gas chromatograph equipped with an automated gas sample valve and a flame ionization detector was used.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 15 days
- Proof of pregnancy: Daily examination of each female for an intravaginal copulation plug or sperm in the vaginal lavage sample.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Days 0–19 of gestation: Females presumed pregnant were housed individually in solid bottom caging with bedding and enrichment. During lactation periods, adult females were housed with their litters. Enrichment was not provided during the approximate period of expected delivery.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each exposure, the vapour concentration was determined by gas chromatography at least once an hour from the test chambers. The control chamber was sampled at least twice a day. Samples of volumes of chamber atmosphere were directly injected into a gas chromatograph (GC) equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30 m x 0.53 mm (nominal diameter) RTX-502.2 fused silica glass column. The vapour concentration was determined from a standard curve derived from gas standards.
Duration of treatment / exposure:
Premating period: 6 hr/day, 5 days/week.
Exposures for P1 males and females were conducted for approximately 2 weeks (TD 0-13). Exposures were then suspended for 2 weeks and subsequently resumed on test day 29. Exposures continued for an approximate 2-week premating period (TD-29-48).

Cohabitation: 6 hr/day, 7 days/week.
Exposures during the cohabitation period were conducted for approximately 2 weeks (TD 49-62).

Gestation: 6 hr/day, 7 days/week.
Females with evidence of mating were exposed during GD 0-19. Gestating dams were not exposed after gestation day 19 or during the approximately 4- day lactation period.

Lactation: No exposure (LD 0-4)

Postcohabitation Exposure: 6 hr/day, 7 days/week.
Males - Exposures continued until the day prior to euthanasia on test days 77-78.
Females with no evidence of copulation – Exposure continued for another 24 days after the end of the cohabitation period.
Details on study schedule:
GESTATION:
Beginning on presumed gestation day 20, female rats were observed at least twice daily for signs of delivery and offspring.

LACTATION:
At each examination period, offspring were individually handled and examined for abnormal behaviour and appearance; any dead or abnormal pups were recorded.
Day 0 (Lactation): Live and dead pups in each litter were counted by sex and individually weighed as soon as possible after delivery was completed.
Day 4 (Litter Sacrifice): Pups in each litter were counted by sex and individually weighed and a gross external examination was performed.

If litters died prior to day 4 of lactation, the dam was sacrificed (unscheduled sacrifice). If the dam died prior to day 4 of lactation, the litter was examined externally, sacrificed, and discarded.
Remarks:
Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals – At least once daily during quarantine and prior to study start, and twice daily thereafter; once daily on exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and at least weekly (except necropsy and FOBs) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: All animals – at least once
Pretest: Weekly
Premating: Twice weekly for the first 2 weeks, and then at least weekly thereafter
Cohabitation: Weekly
Gestation: 0, 7, 14, and 19 G
Lactation: 0, 4 L
Post-cohabitation: Weekly and at Terminal Sacrifice for males and females without evidence of copulation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Calculation of Food Consumption: Weighed each feeder at the beginning and end of interval and subtracted final weight and amount of spillage from the feeder from initial weight
Premating: Weekly
Cohabitation: None
Gestation: 0, 7, 14, and 19 G
Lactation: 0, 4 L

FOOD EFFICIENCY: Yes
Calculation of Mean Daily Food Efficiency: From food consumption and body weight data

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Test Day 48 (all)
Lactation day 4 (pregnant females) (including coagulation)
25 days after the end of cohabitation (non-pregnant females)
Test Days 77-78 (male) (including coagulation)
- Animals fasted: Yes
- How many animals: 10 each sex
- Parameters in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Test Day 48 (all)
Lactation day 4 (pregnant females) (including coagulation)
25 days after the end of cohabitation (non-pregnant females)
Test Days 77-78 (male) (including coagulation)
- Animals fasted: Yes
- How many animals: 10 each sex
- Parameters in Table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1 Neurotoxicity: DI.K1.Neuro.R.D-19695-1422.KD for additional details).
- Time schedule for examinations: during acclimation (baseline) and test days 44-45
- Dose groups that were examined: 6 animals/sex/dose
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation
- open field arena: righting reflex, approach and touch, sharp auditory stimulus, tail pinch
- Chatillon® Digital Force gauge: forelimb grip strength, hindlimb grip strength
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: external alterations, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: Yes
for external and internal abnormalities to the extent possible; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE SCHEDULE:
All surviving P1 rats (females with litters, males, non-pregnant females) - test days 77–86
Two males (2/40) and two females (2/40) died before the scheduled sacrifice

GROSS NECROPSY: Yes (see table No. 4)
All animals. Uterine implantation sites and ovarian corpora lutea were counted in P1 females.

HISTOPATHOLOGY / ORGAN WEIGHTS: The tissues indicated in Table 5 were prepared for microscopic examination and weighed, respectively.
Histopathology - Control and high-dose groups, and all early decedents; (gross lesions in intermediate-dose groups); reproductive organs from all male and female P1 rats that were suspected of impaired reproductive performance (e.g., failure to mate, conceive, sire, or deliver healthy offspring).
Organ Weights - all adults
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND4.
- These animals were subjected to postmortem examinations (macroscopic) as follows: external alterations and euthanized by decapitation. Pups found dead or euthanized in moribund condition underwent a gross examination to the extent possible and were discarded.

GROSS NECROPSY
- Gross necropsy consisted of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Not examined
Statistics:
Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean was used as the experimental unit for statistical evaluation. For each parameter analysed with a trend test the test was applied to the data sequentially. For each parameter analysed with a trend test the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. The level of significance selected was p < 0.05. (See Table 6.)
Reproductive indices:
Refer to Table 7 for reproductive indices calculated.

Offspring viability indices:
Refer to Table 7 for offspring viability indices calculated.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Test substance-related mortality did not occur in any animals. One male in the control group was found dead on test day 71 due to subcutaneous and muscular inflammation of the head. One male in the 50000 ppm group was found dead on test day 32. The cause of death was not determined from the histopathological evaluation. One female in the 50000 ppm group was euthanized on day 50 due to a tail lesion secondary to cage door trauma. One female in the 0 ppm group was found dead on lactation day 0 due to apparent dystocia. No test substance-related clinical signs of toxicity were observed in males or females at any exposure concentration during the premating, gestation, or lactation periods. Low incidences of incidental clinical signs common to the age and strain of rat were occasionally observed.

P1 Animals - Anatomic Pathology: P1 Adults Cause of Death
There were no test substance-related deaths among the P1 adults. Two male and two female P1 adult rats died during the study, however their deaths were considered incidental and not exposure related. One control male was found dead on day 71. The cause of death was subcutaneous and muscular inflammation of the head, probably secondary to trauma. One 50000 ppm male was found dead on day 32. Since there were no relevant microscopic lesions, the cause of death was undetermined. One control female was found dead on day 76; the cause of death was dystocia. One 50000 ppm female was euthanized on day 50 due to a tail lesion secondary to cage door trauma.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods. Weight gain of the 2500 and 10000 ppm males was statistically significantly higher compared to the control group for the interval of test days 43-71. However, this statistical difference was considered to be spurious since a dose-response relationship was not evident. Weight gain for 50000 ppm females was statistically significantly higher compared to the control group during test days 22-29. However, this statistical difference was not considered to be test substance-related since it was transient and was not observed during subsequent intervals. No test substance-related effects or statistically significant differences in food consumption or food efficiency were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods. Food efficiency was statistically significantly lower for 50000 ppm females during test days 36-43, compared to the control group. However, this statistical difference was not considered to be test substance-related since it was transient and did not affect body weight, weight gain, or food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test substance-related effects on the mating index, fertility index, gestation index, precoital interval, gestation length, number of corpora lutea, number of implant sites, percentage of pre-implantation loss, and percentage of post-implantation loss occurred at any exposure concentration. Six pairs of adult males and females did not produce a litter (0, 1, 2, and 3 for the 0, 2500, 10000, and 50000 ppm groups, respectively, including the one female in the 50000 ppm group that was euthanized due to trauma). The fertility index was 100%, 90%, 80%, and 87.5%, respectively. The values for fertility index were within the historical control range of 72.2 to 100% . In addition, gross and microscopic morphological evaluation of reproductive tissues from animals that failed to produce a litter, as well as animals that did produce litters, did not detect a test substance-related morphological change. Therefore, the lower fertility index values for the test substance-exposed groups compared to the concurrent control value of 100% were considered to be within the range of normal biological variation.

The precoital interval for 50000 ppm females was significantly lower compared to the control value. The control group mean (3.5 days) was greater than the mean for historical controls (2.8 days) and was close to the maximum value for the historical control data set of 3.8 days. The 50000 ppm mean was 2.2 days and was within the historical control range of 1.8 to 3.8 days.

P1 Adult Reproductive Failures
There were no test substance-related reproductive failures in this study. Six pairs of P1 adult rats failed to produce a litter. Based on the microscopic evaluation of reproductive organs from males and females, the cause of reproductive failure was diagnosed as undetermined in four pairs (i.e., no relevant lesions), failure to ovulate and mate in one pair, and vaginal ulceration and trauma-related euthanasia in one pair. These causes of reproductive failure were interpreted to be incidental and not exposure related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test substance-related organ weight effects. All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure. In males, all three mean testes weight parameters were decreased in the 2500 ppm group as compared to the control values. The mean relative (% brain weight) testes weight was also decreased in the 10000 ppm group. These were all considered spurious since there was no dose response and no similar finding in the Subchronic Subset animals, including the Subchronic and Subchronic with Recovery Subsets. A statistically significant decrease in the 50000 ppm mean absolute epididymides weight was also interpreted as spurious because there was no microscopic correlate in this subset and no similar finding in the subchronic toxicity subsets. In females, there were no statistically significant differences in any of the mean organ parameters.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross observations in any of the P1 adults. All gross observations, recorded at necropsy, were consistent with normal background lesions in rats of this age and strain.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related microscopic findings in any of the P1 adults. All microscopic findings in these animals were consistent with normal background lesions in rats of this age and strain.

OTHER FINDINGS (PARENTAL ANIMALS)
P1 Animals – Neurobehavioural Evaluations
Forelimb and Hindlimb Grip Strength: No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.
Open Field Observations: No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups. Curled tail observed during the FOB at week 6 in one female in the 10000 ppm group was not considered to be test substance related because the sign did not occur in a dose response fashion.
Motor Activity: No test substance-related effects were observed on motor activity for males or females exposed to any concentration of the test substance. At baseline, the mean number of movements for 2500 ppm females during the total session and for 50000 ppm females during the 3rd interval as well as for the total session was increased (statistically significant) compared to control. Also at baseline, the mean duration of movements for 50000 ppm females during the 5th interval was increased (statistically significant) compared to control. However, these statistical increases were not considered to be test substance related because the animals had not yet been exposed to the test substance. At week 6, the mean duration of movements was decreased during the 3rd interval and increased during the 5th interval (all statistically significant) for 10000 and 50000 ppm males. These statistical differences were not considered to be test substance related and due to biological variation because there was no effect on the total session. The remaining mean values for duration and number of movements for the males and females at week 6 were similar to their respective controls.

P1 Animals - Clinical Pathology
Haematology
There were no exposure-related changes in group mean haematology parameters in male or female rats at any concentration tested. The following statistically significant differences were not considered to be related to exposure to the test substance:
• Mean corpuscular haemoglobin (MCH) was minimally lower in male rats exposed to 50000 ppm (5.3% below the control). There were no statistically significant differences in other haematology parameters in this group, and no exposure-related haematology changes in any males or female subset at any concentration tested. Therefore, the minimally lower MCH in the 50000 ppm males was considered unrelated to exposure and non-adverse.
• Absolute reticulocyte count (ARET) was lower in female rats exposed to 50000 ppm (27% below the control). There were no statistically significant changes in related haematology parameters in female rats in this group and no exposure-related haematology in any males or female subset at any concentration tested. Therefore, the lower ARET in the 50000 ppm female group was considered unrelated to exposure and non-adverse.

Coagulation: There were no statistically significant or exposure-related changes in any group mean coagulation parameter in male or female rats at any concentration tested.

Clinical Chemistry
There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested. The following statistically significant difference was considered unrelated to exposure and nonadverse:
• Creatinine (CREA) was minimally higher in male rats exposed to 50000 ppm (36% above the control). However, there were no statistically significant changes in other kidney-related clinical pathology parameters and no test substance-related microscopic changes in this group. In addition, there were no test substance-related changes in creatinine in any subset of male or female rats at any of the concentrations tested. Therefore, the minimally higher creatine in the 50000 ppm males was considered to be unrelated to exposure and non-adverse.

Urinalyses: There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.

Plasma and Urine Fluoride: There were no statistically significant changes in plasma and urine fluoride parameters in male or female rats.
Dose descriptor:
NOAEC
Effect level:
50 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity based on the absence of effects in all endpoints at the highest concentration tested
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
No test substance-related effects on the number of offspring born, born alive, percentage born alive, number of offspring surviving to lactation day 4, percentage surviving to lactation day 4, offspring sex ratio occurred at any exposure concentration. One litter in the control group was euthanized on lactation day 0 due to the death of one dam in the control group, as a result of dystocia. Delivery for the litter produced by one dam at 50000 ppm commenced on the last exposure day, while the animal was in the exposure chamber. This animal was removed from the chamber, and the delivery proceeded normally.

CLINICAL SIGNS (OFFSPRING)
No test substance-related effects on offspring clinical signs occurred at any exposure concentration.

BODY WEIGHT (OFFSPRING)
No test substance-related effects on offspring body weight occurred at any exposure concentration.

GROSS PATHOLOGY (OFFSPRING)
Five male and 6 female pups were found dead on days 0–5 post-parturition. All 11 pups were necropsied. There were no test substance-related gross observations in these 11 F1 pups.

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
50 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested
Reproductive effects observed:
not specified
Conclusions:
The NOAEC for systemic reproductive effects was 50000 ppm based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The purpose of this study was to determine the potential reproductive toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance.  

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were exposed during an exposure period of approximately 2 weeks during which the animals were exposed 6 hours per day, 5 days per week. Due to a technical issue, exposures for the animals were suspended for 2 weeks. Exposures for animals were resumed on test day 29. Following resumption of exposures, animals were exposed for 6 hours per day, 5 days per week for a 2-week premating period, followed by the cohabitation period of approximately 2 weeks during which exposures were conducted 6 hours per day, 7 days per week. This exposure schedule continued during a gestation period of approximately 3 weeks. Gestating dams were not exposed after Gestation day 19 or during the approximately 4 day lactation period. Females without evidence of mating continued to be exposed for 24 days after the end of the cohabitation period. Exposures for parental males continued until the day prior to sacrifice on test days 77-78. 

 

Concentrations of test substance were generated by metering the test substance into the inhalation chambers and diluting it with conditioned, filtered air, supplemented with oxygen to achieve an oxygen concentration of 20% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography. Temperature, humidity, and airflow were also recorded periodically during each exposure day.  Body weights, clinical signs, and food consumption were recorded throughout the study. Blood and urine samples were collected near the end of the premating period on test day 48 from 5 male and 5 female rats per group. An abbreviated neurobehavioural evaluation was conducted on 6 male and 6 female rats assigned to the Reproduction Subset prior to test substance administration in order to obtain baseline measurements. This neurobehavioural evaluation was conducted again near the end of the pre-mating period for the animals in the Reproduction Subset. Male rats assigned to the Reproduction Subset were euthanized on test days 77-78, selected organs were weighed, and selected tissues were evaluated microscopically.

 

Following the premating period, each female in the Reproduction Subset was paired with a male of the same respective exposure group during an approximately 2 week cohabitation period. Dams were allowed to deliver and rear their offspring until postnatal day 4. Litter examinations (live, dead, or missing pups, individual pup weights and clinical observations) were determined at birth and again on postnatal day 4. Subsequently, lactating females and offspring were sacrificed, selected organs were weighed, and selected tissues were evaluated microscopically. Offspring were evaluated for external abnormalities. 

 

The mean concentrations (± standard error of the mean) were 2500 ± 10, 10000 ± 70, and 51000 ± 650 ppm in chambers targeted at 2500, 10000, and 50000 ppm, respectively. No test substance-related or adverse effects were observed on survival, clinical signs, body weight, weight gain, food consumption, and food efficiency, reproductive performance, or offspring for all animals. There were no adverse, test substance-related effects on neurobehavioural parameters, or clinical pathology parameters. There were no test substance-related effects on organ weight parameters. There were no test substance-related effects on gross or microscopic observations. Under the conditions of the study, the NOAEC for reproductive effects was 50000 ppm based on the absence of effects on reproductive endpoints and offspring at the highest concentration tested. 

Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
282 233 mg/m³
Study duration:
subchronic
Species:
rat
Additional information

Combined repeated dose toxicity study with reproductive/developmental toxicity screening test was conducted in male and female rats according to OECD Guideline 422 at concentrations of 0, 2500, 10000, and 50000 ppm for 6 hours/day, 5 days/week through premating. They were then exposed 6 hours/day, 7 days/week for the cohabitation (males and females) and gestation (females only) periods. No test substance-related adverse effects were observed on reproductive performance, offspring weight, survival, or clinical signs. The NOAEC for reproduction was 50000 ppm based on the absence of effects at the highest concentration tested.


Short description of key information:
Inhalation: OECD 422; rats. NOAEC = 50000 ppm (282233 mg/m3). No test substance-related adverse effects were observed in reproductive parameters examined at the highest concentration tested. Reliability = 1.

Effects on developmental toxicity

Description of key information
Inhalation: OECD 422; rats. NOAEC = 50000 ppm (282233 mg/m3).   No test substance-related adverse effects were observed in maternal or offspring parameters examined at the highest concentration tested. Reliability = 1.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Study included FOB and motor activity endpoints.
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
yes
Remarks:
Study included FOB and motor activity endpoints
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No
- Housing:
Non-Exposure Periods: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation, gestation/lactation as described below .
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
Gestation/Lactation: Females presumed pregnant were housed individually in solid bottom caging with bedding and enrichment. During lactation periods, adult females were housed with their litters. Enrichment was not provided during the approximate period of expected delivery.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: filtered and conditioned air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style) with a nominal internal volume of 350 L
- Method of holding animals in test chamber: individually placed in stainless steel wire-mesh modules (one/module) and exposed, whole-body, inside the exposure chamber; during the mating period animals were housed as mating pairs and exposed in the same manner.
- Source and rate of air: filtered and conditioned air and oxygen
- System of generating vapour:test substance vapour and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 19–24°C; 41–82%; pressure not reported
- Air flow rate: 70–114 L/min
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: to fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by gas chromatography at approximately 30-minute intervals in the test chambers. A gas chromatograph equipped with an automated gas sample valve and a flame ionization detector was used.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each exposure, the vapour concentration was determined by gas chromatography at least once an hour from the test chambers. The control chamber was sampled at least twice a day. Samples of volumes of chamber atmosphere were directly injected into a gas chromatograph (GC) equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30 m x 0.53 mm (nominal diameter) RTX-502.2 fused silica glass column. The vapour concentration was determined from a standard curve derived from gas standards.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 15 days
- Proof of pregnancy: Daily examination of each female for an intravaginal copulation plug or sperm in the vaginal lavage sample.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Days 0–19 of gestation: Females presumed pregnant were housed individually in solid bottom caging with bedding and enrichment. During lactation periods, adult females were housed with their litters. Enrichment was not provided during the approximate period of expected delivery.
Duration of treatment / exposure:
Premating period: 6 hr/day, 5 days/week.
Exposures for P1 males and females were conducted for approximately 2 weeks (TD 0-13). Exposures were then suspended for 2 weeks and subsequently resumed on test day 29. Exposures continued for an approximate 2-week premating period (TD-29-48).

Cohabitation: 6 hr/day, 7 days/week.
Exposures during the cohabitation period were conducted for approximately 2 weeks (TD 49-62).

Gestation: 6 hr/day, 7 days/week.
Females with evidence of mating were exposed during GD 0-19. Gestating dams were not exposed after gestation day 19 or during the approximately 4- day lactation period.

Lactation: No exposure (LD 0-4)

Postcohabitation Exposure: 6 hr/day, 7 days/week.
Males - Exposures continued until the day prior to euthanasia on test days 77-78.
Females with no evidence of copulation – Exposure continued for another 24 days after the end of the cohabitation period.
Duration of test:
90 days
Remarks:
Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals – At least once daily during quarantine and prior to study start, and twice daily thereafter; once daily on exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and at least weekly (except necropsy and FOBs) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: All animals – at least once
Pretest: Weekly
Premating: Twice weekly for the first 2 weeks, and then at least weekly thereafter
Cohabitation: Weekly
Gestation: 0, 7, 14, and 19 G
Lactation: 0, 4 L
Post-cohabitation: Weekly and at Terminal Sacrifice for males and females without evidence of copulation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Calculation of Food Consumption: Weighed each feeder at the beginning and end of interval and subtracted final weight and amount of spillage from the feeder from initial weight
Premating: Weekly
Cohabitation: None
Gestation: 0, 7, 14, and 19 G
Lactation: 0, 4 L

FOOD EFFICIENCY: Yes
Calculation of Mean Daily Food Efficiency: From food consumption and body weight data

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Test Day 48 (all)
Lactation day 4 (pregnant females) (including coagulation)
25 days after the end of cohabitation (non-pregnant females)
Test Days 77-78 (male) (including coagulation)
- Animals fasted: Yes
- How many animals: 10 each sex
- Parameters in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Test Day 48 (all)
Lactation day 4 (pregnant females) (including coagulation)
25 days after the end of cohabitation (non-pregnant females)
Test Days 77-78 (male) (including coagulation)
- Animals fasted: Yes
- How many animals: 10 each sex
- Parameters in Table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1 Neurotoxicity: DI.K1.Neuro.R.D-19695-1422.KD for additional details).
- Time schedule for examinations: during acclimation (baseline) and test days 44-45
- Dose groups that were examined: 6 animals/sex/dose
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation
- open field arena: righting reflex, approach and touch, sharp auditory stimulus, tail pinch
- Chatillon® Digital Force gauge: forelimb grip strength, hindlimb grip strength
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

On lactation days 0 and 4, offspring were individually handled and examined for abnormal behaviour and appearance; any dead or abnormal pups were recorded. On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the dam was sacrificed. If the dam died prior to Day 4 of lactation, the litter was examined externally and sacrificed. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation.

Gross Examination of Dead Pups: Yes, for external abnormalities; possible cause of death was determined for pups born or found dead.
Statistics:
Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean was used as the experimental unit for statistical evaluation. For each parameter analysed with a trend test the test was applied to the data sequentially. For each parameter analysed with a trend test the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. The level of significance selected was p < 0.05. (See Table 6.)
Indices:
Refer to Table 7 for reproductive indices calculated.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Test substance-related mortality did not occur in any animals. One male in the control group was found dead on test day 71 due to subcutaneous and muscular inflammation of the head. One male in the 50000 ppm group was found dead on test day 32. The cause of death was not determined from the histopathological evaluation. One female in the 50000 ppm group was euthanized on day 50 due to a tail lesion secondary to cage door trauma. One female in the 0 ppm group was found dead on lactation day 0 due to apparent dystocia. No test substance-related clinical signs of toxicity were observed in males or females at any exposure concentration during the premating, gestation, or lactation periods. Low incidences of incidental clinical signs common to the age and strain of rat were occasionally observed.

P1 Animals - Anatomic Pathology: P1 Adults Cause of Death
There were no test substance-related deaths among the P1 adults. Two male and two female P1 adult rats died during the study, however their deaths were considered incidental and not exposure related. One control male was found dead on day 71. The cause of death was subcutaneous and muscular inflammation of the head, probably secondary to trauma. One 50000 ppm male was found dead on day 32. Since there were no relevant microscopic lesions, the cause of death was undetermined. One control female was found dead on day 76; the cause of death was dystocia. One 50000 ppm female was euthanized on day 50 due to a tail lesion secondary to cage door trauma.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods. Weight gain of the 2500 and 10000 ppm males was statistically significantly higher compared to the control group for the interval of test days 43-71. However, this statistical difference was considered to be spurious since a dose-response relationship was not evident. Weight gain for 50000 ppm females was statistically significantly higher compared to the control group during test days 22-29. However, this statistical difference was not considered to be test substance-related since it was transient and was not observed during subsequent intervals. No test substance-related effects or statistically significant differences in food consumption or food efficiency were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods. Food efficiency was statistically significantly lower for 50000 ppm females during test days 36-43, compared to the control group. However, this statistical difference was not considered to be test substance-related since it was transient and did not affect body weight, weight gain, or food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test substance-related effects on the mating index, fertility index, gestation index, precoital interval, gestation length, number of corpora lutea, number of implant sites, percentage of pre-implantation loss, and percentage of post-implantation loss occurred at any exposure concentration. Six pairs of adult males and females did not produce a litter (0, 1, 2, and 3 for the 0, 2500, 10000, and 50000 ppm groups, respectively, including the one female in the 50000 ppm group that was euthanized due to trauma). The fertility index was 100%, 90%, 80%, and 87.5%, respectively. The values for fertility index were within the historical control range of 72.2 to 100% . In addition, gross and microscopic morphological evaluation of reproductive tissues from animals that failed to produce a litter, as well as animals that did produce litters, did not detect a test substance-related morphological change. Therefore, the lower fertility index values for the test substance-exposed groups compared to the concurrent control value of 100% were considered to be within the range of normal biological variation.

The precoital interval for 50000 ppm females was significantly lower compared to the control value. The control group mean (3.5 days) was greater than the mean for historical controls (2.8 days) and was close to the maximum value for the historical control data set of 3.8 days. The 50000 ppm mean was 2.2 days and was within the historical control range of 1.8 to 3.8 days.

P1 Adult Reproductive Failures
There were no test substance-related reproductive failures in this study. Six pairs of P1 adult rats failed to produce a litter. Based on the microscopic evaluation of reproductive organs from males and females, the cause of reproductive failure was diagnosed as undetermined in four pairs (i.e., no relevant lesions), failure to ovulate and mate in one pair, and vaginal ulceration and trauma-related euthanasia in one pair. These causes of reproductive failure were interpreted to be incidental and not exposure related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test substance-related organ weight effects. All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure. In males, all three mean testes weight parameters were decreased in the 2500 ppm group as compared to the control values. The mean relative (% brain weight) testes weight was also decreased in the 10000 ppm group. These were all considered spurious since there was no dose response and no similar finding in the Subchronic Subset animals, including the Subchronic and Subchronic with Recovery Subsets. A statistically significant decrease in the 50000 ppm mean absolute epididymides weight was also interpreted as spurious because there was no microscopic correlate in this subset and no similar finding in the subchronic toxicity subsets. In females, there were no statistically significant differences in any of the mean organ parameters.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross observations in any of the P1 adults. All gross observations, recorded at necropsy, were consistent with normal background lesions in rats of this age and strain.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related microscopic findings in any of the P1 adults. All microscopic findings in these animals were consistent with normal background lesions in rats of this age and strain.

OTHER FINDINGS (PARENTAL ANIMALS)
P1 Animals – Neurobehavioural Evaluations
Forelimb and Hindlimb Grip Strength: No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.
Open Field Observations: No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups. Curled tail observed during the FOB at week 6 in one female in the 10000 ppm group was not considered to be test substance related because the sign did not occur in a dose response fashion.
Motor Activity: No test substance-related effects were observed on motor activity for males or females exposed to any concentration of the test substance. At baseline, the mean number of movements for 2500 ppm females during the total session and for 50000 ppm females during the 3rd interval as well as for the total session was increased (statistically significant) compared to control. Also at baseline, the mean duration of movements for 50000 ppm females during the 5th interval was increased (statistically significant) compared to control. However, these statistical increases were not considered to be test substance related because the animals had not yet been exposed to the test substance. At week 6, the mean duration of movements was decreased during the 3rd interval and increased during the 5th interval (all statistically significant) for 10000 and 50000 ppm males. These statistical differences were not considered to be test substance related and due to biological variation because there was no effect on the total session. The remaining mean values for duration and number of movements for the males and females at week 6 were similar to their respective controls.

P1 Animals - Clinical Pathology
Haematology
There were no exposure-related changes in group mean haematology parameters in male or female rats at any concentration tested. The following statistically significant differences were not considered to be related to exposure to the test substance:
• Mean corpuscular haemoglobin (MCH) was minimally lower in male rats exposed to 50000 ppm (5.3% below the control). There were no statistically significant differences in other haematology parameters in this group, and no exposure-related haematology changes in any males or female subset at any concentration tested. Therefore, the minimally lower MCH in the 50000 ppm males was considered unrelated to exposure and non-adverse.
• Absolute reticulocyte count (ARET) was lower in female rats exposed to 50000 ppm (27% below the control). There were no statistically significant changes in related haematology parameters in female rats in this group and no exposure-related haematology in any males or female subset at any concentration tested. Therefore, the lower ARET in the 50000 ppm female group was considered unrelated to exposure and non-adverse.

Coagulation: There were no statistically significant or exposure-related changes in any group mean coagulation parameter in male or female rats at any concentration tested.

Clinical Chemistry
There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested. The following statistically significant difference was considered unrelated to exposure and nonadverse:
• Creatinine (CREA) was minimally higher in male rats exposed to 50000 ppm (36% above the control). However, there were no statistically significant changes in other kidney-related clinical pathology parameters and no test substance-related microscopic changes in this group. In addition, there were no test substance-related changes in creatinine in any subset of male or female rats at any of the concentrations tested. Therefore, the minimally higher creatine in the 50000 ppm males was considered to be unrelated to exposure and non-adverse.

Urinalyses: There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.

Plasma and Urine Fluoride: There were no statistically significant changes in plasma and urine fluoride parameters in male or female rats.
Dose descriptor:
NOAEC
Effect level:
50 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
50 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
No test substance-related effects on the number of offspring born, born alive, percentage born alive, number of offspring surviving to lactation day 4, percentage surviving to lactation day 4, offspring sex ratio occurred at any exposure concentration. One litter in the control group was euthanized on lactation day 0 due to the death of one dam in the control group, as a result of dystocia. Delivery for the litter produced by one dam at 50000 ppm commenced on the last exposure day, while the animal was in the exposure chamber. This animal was removed from the chamber, and the delivery proceeded normally.

CLINICAL SIGNS (OFFSPRING)
No test substance-related effects on offspring clinical signs occurred at any exposure concentration.

BODY WEIGHT (OFFSPRING)
No test substance-related effects on offspring body weight occurred at any exposure concentration.

GROSS PATHOLOGY (OFFSPRING)
Five male and 6 female pups were found dead on days 0–5 post-parturition. All 11 pups were necropsied. There were no test substance-related gross observations in these 11 F1 pups.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The NOAEL for reproductive effects was 50000 ppm based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

The purpose of this study was to determine the potential reproductive toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance.  

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were exposed during an exposure period of approximately 2 weeks during which the animals were exposed 6 hours per day, 5 days per week. Due to a technical issue, exposures for the animals were suspended for 2 weeks. Exposures for animals were resumed on test day 29. Following resumption of exposures, animals were exposed for 6 hours per day, 5 days per week for a 2-week premating period, followed by the cohabitation period of approximately 2 weeks during which exposures were conducted 6 hours per day, 7 days per week. This exposure schedule continued during a gestation period of approximately 3 weeks. Gestating dams were not exposed after Gestation day 19 or during the approximately 4 day lactation period. Females without evidence of mating continued to be exposed for 24 days after the end of the cohabitation period. Exposures for parental males continued until the day prior to sacrifice on test days 77-78. 

 

Concentrations of test substance were generated by metering the test substance into the inhalation chambers and diluting it with conditioned, filtered air, supplemented with oxygen to achieve an oxygen concentration of 20% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography. Temperature, humidity, and airflow were also recorded periodically during each exposure day.  Body weights, clinical signs, and food consumption were recorded throughout the study. Blood and urine samples were collected near the end of the premating period on test day 48 from 5 male and 5 female rats per group. An abbreviated neurobehavioural evaluation was conducted on 6 male and 6 female rats assigned to the Reproduction Subset prior to test substance administration in order to obtain baseline measurements. This neurobehavioural evaluation was conducted again near the end of the pre-mating period for the animals in the Reproduction Subset. Male rats assigned to the Reproduction Subset were euthanized on test days 77-78, selected organs were weighed, and selected tissues were evaluated microscopically.

 

Following the premating period, each female in the Reproduction Subset was paired with a male of the same respective exposure group during an approximately 2 week cohabitation period. Dams were allowed to deliver and rear their offspring until postnatal day 4. Litter examinations (live, dead, or missing pups, individual pup weights and clinical observations) were determined at birth and again on postnatal day 4. Subsequently, lactating females and offspring were sacrificed, selected organs were weighed, and selected tissues were evaluated microscopically. Offspring were evaluated for external abnormalities. 

 

The mean concentrations (± standard error of the mean) were 2500 ± 10, 10000 ± 70, and 51000 ± 650 ppm in chambers targeted at 2500, 10000, and 50000 ppm, respectively. No test substance-related or adverse effects were observed on survival, clinical signs, body weight, weight gain, food consumption, and food efficiency, reproductive performance, or offspring for all animals. There were no adverse, test substance-related effects on neurobehavioural parameters, or clinical pathology parameters. There were no test substance-related effects on organ weight parameters. There were no test substance-related effects on gross or microscopic observations. Under the conditions of the study, the NOAEL for reproductive effects was 50000 ppm based on the absence of effects on reproductive endpoints and offspring at the highest concentration tested. 

Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
282 233 mg/m³
Study duration:
subchronic
Species:
rat
Additional information

A combined repeated dose toxicity study with reproductive/developmental toxicity screening test was conducted in male and female rats according to OECD Guideline 422 at concentrations of 0, 2500, 10000, and 50000 ppm for 6 hours/day, 5 days/week through premating. They were then exposed 6 hours/day, 7 days/week for the cohabitation (males and females) and gestation (females only) periods. No test substance-related adverse effects were observed on reproductive performance, offspring weight, survival, or clinical signs. The NOAEC for offspring was 50000 ppm based on the absence of effects at the highest concentration tested.

Justification for classification or non-classification

The test substance did not adversely affect reproductive organs, reproductive function, or foetal development in a repeat dose/developmental/reproductive toxicity screening study in rats. Therefore, the substance does not need to be classified for reproductive or developmental toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.