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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. Although data provided have a report year after 2009, the study was performed to fulfill needs required by other governmental registrations and/or product stewardship purposes. This study was not performed to specifically fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
number of animals varied
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
number of animals varied
Qualifier:
according to
Guideline:
other: Japanese MAFF 12 Nousan No. 8147, Guidelines Related to the Study Report for the Registration Application of Pesticides, 2000
Deviations:
yes
Remarks:
number of animals varied
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
number of animals varied
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.9%.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 6 months [Hra:(NZW)SPF]
- Weight at study initiation: 2900 g to 3975 g on gestation day 0
- Fasting period before study: No
- Housing: housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®
- Diet: PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322, ad libitum, ecxept during exposures
- Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum, except during exposures

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6°C to 19.8°C
- Humidity (%): 43.8% to 63.5%
- Air changes 10 fresh air changes per hour
- Photoperiod: 12 light/12 dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
Test substance exposures were conducted in four 1.5-m3 stainless-steel and glass exposure chambers and the control group was exposed in a 2.0-m3 stainless-steel and glass exposure chamber. Chamber supply air was provided from a high-efficiency particulate air (HEPA) filter and an activated charcoal bed. Treatment of exhaust air consisted of charcoal and HEPA filtration. Vapours of the test substance were generated by releasing the test substance in gas form from the original cylinders using a Brisk Heat blanket controlled by a J-type thermocouple and an Omega® temperature controller set to approximately 60°C to 70°C. For Phase I of the study, 1 cylinder was used to generate test substance atmospheres for Chambers 2 and 5 and a second cylinder was used to generate test substance atmospheres for Chambers 3 and 4. For Chambers 2 and 5, test substance was delivered to a manifold system and was monitored using Top Trak Mass Flow Meters and controlled using flow control valves. For Chambers 3 and 4, test substance was delivered to a manifold system and was monitored using Omega® digital mass flowmeters and controlled using flow control valves. For Phase II of the study, 1 cylinder was used to generate test substance atmospheres for Chambers 2, 3, and 4. Test substance was delivered to 1 manifold system. For Chamber 2, test substance was monitored using a Sierra Top Trak Mass Flowmeter and was controlled using a flow control valve. For chambers 3 and 4, test substance was monitored using Omega® digital mass flowmeters and controlled using flow control valves. To aid in vapour delivery to the manifold, a coalescing trap was placed in-line between the cylinder regulator and manifold to prevent liquid test substance from reaching the manifold system. For the test substance delivery systems, a dilution source of compressed air was added in-line at a ‘T’ fitting to aid in test substance vaporization. Compressed air was controlled using a coilhose pneumatics regulator. All test substance delivery lines from the cylinder to the manifold and including the manifold were ¼-inch stainless steel and were heated to approximately 100°C (Phase I) and 90°C (Phase II) using Omega® heat tapes, temperature controllers, and J-type thermocouples. Delivery lines from the flow controllers were ¼-inch stainless steel and were heated to approximately 60°C using Omega® heat tapes, temperature controllers, and J-type thermocouples. Test substance vapours mixed with supply air at the chamber inlet.

TEST ATMOSPHERE:
Analysed exposure concentrations were determined approximately every 45 minutes using a gas chromatograph (GC). Samples were collected from the approximate animal breathing zone of the inhalation exposure chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of Analysis: GC
GC: Hewlett Packard 5890 Series II
GC Detector: FID
GC Column: Agilent, DB-5, 35 m × 1.50 mm ID, 0.5-micron film thickness
Integrator: Agilent 3396 Series III/Hewlett Packard Model 3396A

Homogeneity of exposure concentrations for Chambers 2 and 5 were evaluated during the method development phase of the study prior to Phase 1 of animal exposure. Homogeneity determination for Chambers 3 and 4 were performed after Phase 1 of animal exposure but before Phase II of animal exposure during method development. Four test locations (Lower Front, Upper Front, Upper Rear, and Lower Rear) and a reference location were used for these determinations. Samples were collected and analysed on the GC as rapidly as possible by alternating from the reference location and then to a test location. Homogeneity was performed in triplicate for each test substance exposure chamber. The measured concentration was calculated as a percent difference for each position from the reference location. Homogeneity of the exposure atmospheres were considered acceptable if the mean percent variation was within ± 10% for each test location.


Concentration verification (% of Target))
Phase 1
Target Concentration (ppm): 0, 2500, 5000, 7500, 15000
Mean Concentration (ppm): 0, 2426, 4934, 7429, 14667

Phase 2
Target Concentration (ppm): 0, 2500, 5000, 7500
Mean Concentration (ppm): 0, 2415, 5047, 7305
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation Days 7 to 28
Frequency of treatment:
6 hours daily
Duration of test:
29 days
No. of animals per sex per dose:
24 females for doses 0, 2500, 5000 and 7500 ppm (Groups 1 thru 4 respectively)
12 females for dose 15000 ppm (Group 5)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Exposure levels were selected based on the results of previous studies. Reduced maternal body weight gain, food consumption, and foetal body weight was observed at 10000 ppm in a prenatal rat developmental study where female rats were exposed to the test substance for 6 hours per day during gestation days 6-20, inclusive. No test substance-related adverse effects were noted at concentrations up to and including 1500 ppm. In a 90-day repeated-dose study in rats test substance-related adverse effects were observed in males at concentrations greater than or equal to 7500 ppm and in females at 10000 ppm and consisted of reduced food consumption and reduced body weight gain. The highest exposure level in the current study was chosen with the aim to induce some developmental and/or maternal toxicity but not death or severe suffering. Other exposure levels were selected to produce a gradation of toxic effects, if any are observed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to exposure during the treatment period). At the approximate midpoint of exposure, animals visible through the chamber windows were observed for mortality and overt clinical signs of toxicity. Animals were also observed for signs of toxicity at approximately 1 hour following exposure; the absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 (by supplier under non-GLP conditions), 4, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-20, 20-29, and 7-29. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.

FOOD CONSUMPTION: Yes
- Food consumption: Individual food consumption was recorded on gestation days 4-29.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: A gross necropsy was performed on females that were euthanized in extremis during the course of the study. The number and location of implantation sites, corpora lutea, and viable foetuses were recorded. All surviving rabbits were euthanized on gestation day 29. The uterus and ovaries were then exposed and excised. Heart, skeletal muscle (rectus femoris), kidney (2), diaphragm, and gross lesions were collected from all maternal animals and placed into 10% neutral-buffered formalin for possible future histopathological examination and archived. Microscopic examination was performed on the heart from all females on study.
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: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes [all per litter]
Statistics:
Statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable foetuses, and foetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable foetuses, early and late resorptions, total resorptions, pre- and post implantation loss, and foetal sex distribution), total foetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Test substance-related moribundity was noted for females in the 7500 and 15000 ppm groups. In the 15000 ppm group, 7 of 12 females in Phase 1 were euthanized in extremis during gestation days 18-27 due to clinical findings of impaired use of the hind limbs (7 females), increased respiration (3 females), hypoactivity (3 females), tonic convulsions (2 females), laboured respiration (2 females), prostration (1 female), and/or a pale body (1 female). In addition, 3 of the females with impaired use of the hind limbs were noted with a dislocation of the lumbar vertebrae at necropsy; this was likely due to struggling to use the hind limbs. Due to this excessive toxicity, the exposure level of 15000 ppm was not evaluated during Phase 2. In the 7500 ppm group, 1 female was euthanized in extremis on gestation day 25 due to clinical findings of impaired use of the hind limbs, tonic convulsions, and rapid and shallow respiration. No other test substance-related moribundity or mortality was noted. Clinical findings of decreased defecation were also noted for females at these exposure levels.

Test substance-related mean body weight losses or reduced mean body weight gains, with corresponding reduced mean food consumption, were noted for females in the 15000 ppm group generally throughout the exposure period (gestation days 7-29). As a result, mean body weights in this group were up to 7.6% lower than the control group. In addition, mean net body weight change in this group was lower than the control group. These results were considered test substance-related and adverse. Reduced mean body weight gains were noted in the 2500, 5000, and 7500 ppm groups when the entire exposure period was evaluated, with corresponding effects on mean food consumption noted in the 5000 and 7500 ppm groups during gestation days 13-20. However, mean body weights in these groups were not affected. Therefore, these results were considered test substance-related but not adverse. Mean net body weights, net body weight changes, and gravid uterine weights in the 2500, 5000, and 7500 ppm groups were similar to the control group.

No test substance-related macroscopic findings were noted at necropsy for any females at the scheduled gestation day 29 necropsy. There were no test substance-related microscopic findings noted in the heart at any exposure level.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
5 000 other: ppm (nominal) (33548 mg/m3)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
7 500 other: ppm (nominal) (50322 mg/m3)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Test substance-related lower (15.2% to 20.9%) mean foetal weights were noted in the 15000 ppm group (only 4 litters available for evaluation). Mean foetal weights in the 2500, 5000, and 7500 ppm groups were unaffected by test substance exposure. Although mean foetal weights in the 2500, 5000, and 7500 ppm groups were lower (up to 11.2%) than the concurrent control group, the results were within the ranges of values in the test facility historical control data (36.6 g to 44.8 g for males, 35.9 g to 44.4 g for females, and 36.4 g to 44.6 g for males and females combined) and were influenced by 1-2 litters in each of these groups with mean foetal weights below 30.0 g. Therefore, the differences were not considered test substance-related. Intrauterine survival and foetal morphology at all exposure levels were unaffected by test substance exposure.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
7 500 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity lower fetal weights at 15000 ppm (100644 mg/m3)
Remarks on result:
other: see remarks
Remarks:
(50322 mg/m3)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
NOAEC maternal toxicity = 5000 ppm
NOAEC developmental toxicity = 7500 ppm
Executive summary:

The objectives of this study were to determine the potential of the test item to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed adverse-effect concentration (NOAEC) for maternal and developmental toxicity. The test substance was administered via whole body inhalation exposure to 4 groups of time-mated female New Zealand White rabbits for 6 hours daily, from gestation days 7 through 28. Nominal exposure levels were 0, 2500, 5000, 7500, and 15000 ppm. There were 12 animals in the highest exposure group, 24 in all others.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 29, a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Test substance-related moribundity was noted for females in the 7500 and 15000 ppm groups. In the 15000 ppm group, 7 of 12 females in Phase 1 were euthanized in extremis during gestation days 18-27 due to clinical findings of impaired use of the hind limbs (7 females), increased respiration (3 females), hypoactivity (3 females), tonic convulsions (2 females), laboured respiration (2 females), prostration (1 female), and/or a pale body (1 female). In addition, 3 of the females with impaired use of the hind limbs were noted with a dislocation of the lumbar vertebrae at necropsy; this was likely due to struggling to use the hind limbs. Due to this excessive toxicity, the exposure level of 15000 ppm was not evaluated during Phase 2. In the 7500 ppm group, 1 female was euthanized in extremis on gestation day 25 due to clinical findings of impaired use of the hind limbs, tonic convulsions, and rapid and shallow respiration. No other test substance-related moribundity or mortality was noted. Clinical findings of decreased defecation were also noted for females at these exposure levels. Test substance-related mean body weight losses or reduced mean body weight gains, with corresponding reduced mean food consumption, were noted for females in the 15000 ppm group generally throughout the exposure period (gestation days 7-29). As a result, mean body weights in this group were up to 7.6% lower than the control group. In addition, mean net body weight change in this group was lower than the control group. These results were considered test substance-related and adverse. Reduced mean body weight gains were noted in the 2500, 5000, and 7500 ppm groups when the entire exposure period was evaluated, with corresponding effects on mean food consumption noted in the 5000 and 7500 ppm groups during gestation days 13-20. However, mean body weights in these groups were not affected. Therefore, these results were considered test substance-related but not adverse. Mean net body weights, net body weight changes, and gravid uterine weights in the 2500, 5000, and 7500 ppm groups were similar to the control group. No test substance-related macroscopic findings were noted at necropsy for any females at the scheduled gestation day 29 necropsy.

Test substance-related lower (15.2% to 20.9%) mean foetal weights were noted in the 15000 ppm group (only 4 litters available for evaluation). Mean foetal weights in the 2500, 5000, and 7500 ppm groups were unaffected by test substance exposure. Although mean foetal weights in the 2500, 5000, and 7500 ppm groups were lower (up to 11.2%) than the concurrent control group, the results were within the ranges of values in the test facility historical control data (36.6 g to 44.8 g for males, 35.9 g to 44.4 g for females, and 36.4 g to 44.6 g for males and females combined) and were influenced by 1-2 litters in each of these groups with mean foetal weights below 30.0 g. Therefore, the differences were not considered test substance-related. Intrauterine survival and foetal morphology at all exposure levels were unaffected by test substance exposure.

Based on adverse clinical findings and moribundity of maternal females at 7500 and 15000 ppm and reduced mean body weights and food consumption at 15000 ppm, an exposure level of 5000 ppm was considered to be the no-observed-adverse-effect level (NOAEC) for maternal toxicity. Due to the lower foetal weights at 15000 ppm (only 4 litters evaluated) an exposure level of 7500 ppm was considered to be the NOAEC for prenatal development when the test item was administered via whole body inhalation for 6 hours daily to time-mated New Zealand White rabbits.