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Toxicological information

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
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Analytical purity: 99.99%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 55 days old, approximately
- Weight at study initiation: Male Rats – 234.9-284.4 grams/Female Rats – 170.2-229.1 grams
- Fasting period before study: None
- Housing: Stainless steel, wire-mesh cages suspended above cageboards, individually, for male and female rats during non-mating periods and as breeding pairs during mating. Polycarbonate pans with bedding, individually, for females with evidence of copulation and with litters
- Diet (e.g. ad libitum): Ad libitum at all times other than during exposure
- Water (e.g. ad libitum): Ad libitum at all times other than during exposure
- Acclimation period: Quarantined for 9 days of 12-day pretest period

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

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: Nitrogen and Filtered air
Details on exposure:
- Exposure apparatus: NYU type stainless steel, Teflon, or glass; an NYU type 750L chamber was used for test substance exposures and an NYU type 1000L chamber was used for control (air only) exposures The exposure chambers were cubical with square-pyramidal chamber inlets and outlets with a tangential feed at the chamber inlet which promotes uniform chamber distribution of the test substance. The chamber volume was chosen so that the total body volume of the test animals was not expected to exceed approximately 5% of the chamber volume.
- Source and rate of air: Houseline, filtered air
- Method of conditioning air: Heated, round-bottom, flash evaporation flasks
- System of generating vapour atmospheres: Flash evaporation of the test substance in air. The test substance was metered to heated round-bottom, flash evaporation flasks. For the low level, the test substance was metered with a syringe infusion pump. For the intermediate and high level, the test substance was metered with pumps equipped with pistons. Houseline air was metered to the round-bottom flasks by mass flow controllers and swept the test substance vapour to glass and stainless steel transfer tubes that connected the heated flasks to the top of the chamber turrets. Filtered air entered the chamber turret tangentially and mixed with test substance vapour inside the turret. A stainless steel baffle was positioned beneath the turret to promote uniform distribution of the chamber atmosphere. Chamber concentrations of test substance were controlled by setting the test substance feed rate to the round-bottom flask. The control chamber had the same type generation system except there was no infusion pump or Masterflex pump used. Generation air, the infusion pump, Masterflex pumps, and heating mantles were all monitored and controlled by a customized Camile Inhalation Toxicology Automated Data System (CITADS).
- Temperature, humidity, pressure in air chamber: 19-25°C, 30-70%. Pressure not reported.
- Air flow rate: Not reported
- Air change rate: At least 10 air changes/hour
- Treatment of exhaust air: Test atmospheres were discharged directly into a dedicated exhaust fan and exhaust stack.
Details on mating procedure:
Animals were paired for mating after approximately 10 weeks of exposure to the test substance. Each female was continually housed on a 1:1 basis with a randomly selected, nonsibling male of the same concentration level, in the male's cage. Animals remained paired until evidence of mating was observed or until a period of 2 weeks elapsed. Once daily, prior to placing the animals in the exposure chambers, each female was examined for evidence of copulation/mating (intravaginal copulation plug in situ or sperm in vaginal lavage sample). The day evidence of mating was observed is designated gestation day (GD) 0. On the day copulation was confirmed, the female was transferred back to individual cage housing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During each exposure, the atmospheric concentration of the test substance was determined at least hourly by gas chromatography. Samples of chamber atmosphere were drawn from the breathing zone of the animals and were directly injected into a gas chromatograph (GC) equipped with a gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 110°C on a fused silica glass column. The atmospheric concentration of the test substance was determined from a standard curve derived from gas standards. Standards were prepared prior to the exposure by injecting known volumes of the liquid test substance into Tedlar bags containing known volumes of air.
Duration of treatment / exposure:
107 days, approximately, for males
121 days, approximately, for females
Frequency of treatment:
6 hours/day for 5 days/week during premating for approximately 10 weeks for males and females,
6 hours/day for 7 days/week as specified below:
Cohabitation period for males/females – for approximately 14 days
Postcohabitation for males – for approximately 23 days
When evidence of copulation was observed, the gestation period was initiated
Gestation for females – for approximately 20 days
Lactation for females – for approximately 17 days
Offspring were not exposed in the exposure chamber
Postcohabitation for females – for approximately 20 days
Females without evidence of copulation were exposed for 20 days or until evidence of delivery/impending delivery.

Doses / concentrations
Remarks:
Doses / Concentrations:
Males/Females: 0, 500, 2000, 3500 ppm (0; 5154; 20618; 36081 mg/m3, respectively)
Basis:
nominal conc.
No. of animals per sex per dose:
25 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous 90-day inhalation study, rats were exposed for 6 hours per day, 5 days per week to 0, 500, 2000, or 3500 ppm of test substance. Clinical signs of convulsions, tremors, abnormal gait, and jerky leg movements occurred during exposure to 2000 or 3500 ppm. None of these effects were observed during non-exposure periods. Decreased motor activity was observed after 8 and 13 weeks of exposure to 3500 ppm. Decreased serum albumin also occurred in rats exposed to 3500 ppm. There were no effects on body weight, food consumption, organ weights, or tissue morphology. In a previous rat developmental study, presumed pregnant females were exposed on gestation days 7 – 16 at concentrations of 0, 500, 2000, or 3500 ppm. Clinical signs of convulsions and tremors were observed during exposure at 2000 and 3500 ppm. Increased incidences of stained fur also occurred at 2000 ppm and above. Mean foetal body weight was decreased in the 3500 ppm group. There were no effects on maternal weight gain, food consumption, or the incidence of foetal abnormalities. Based on the effects observed in these studies, the exposure concentrations selected for this study were 0, 500, 2000, and 3500 ppm.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
EXAMINATIONS
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; after being transferred into polycarbonate pans, females were observed at least twice daily for signs of delivery and offspring, until delivery was complete

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

CLINICAL OBSERVATIONS DURING EXPOSURES: Yes
- Time schedule: 3 times during each exposure

BODY WEIGHT: Yes
- Time schedule for examinations:
Premating, Cohabitation - Weekly
Post-cohabitation for males and females that were not pregnant or presumed not pregnant – Weekly
Gestation – Gestation Days 0, 7, 14, 21
Lactation – Lactation Days 0, 7, 14, 21
Scheduled Sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations:
Premating - Weekly
Cohabitation - None
Post-cohabitation for males and females that were not pregnant or presumed not pregnant – None
Gestation – Gestation Days 0, 7, 14, 21
Lactation – Lactation Days 0, 7, 14
Estrous cyclicity (parental animals):
Not evaluated
Sperm parameters (parental animals):
Not evaluated
Litter observations:
Litter Evaluations - Each litter was examined as soon as possible after delivery was completed. The day when delivery was complete was designated lactation day (LD) 0 for dams and postnatal day (PND) 0 for pups. On LD 0, 4, 7, 14, and 21, live and dead pups in each litter were counted by sex and live pups were individually weighed. Each live pup was individually handled and examined for abnormal behaviour and an external examination was conducted. If a litter died prior to LD 21, the dam was sacrificed. If the dam died prior to LD 21, the litter was sacrificed.
Culling - On LD 4, after litter observations were performed and body weights were collected, litters with more than 8 pups were reduced to 8 pups/litter (4/sex, when possible). The method of selecting the culled pups was random. Culled pups were euthanized by decapitation and discarded. Culled pups with grossly observable abnormalities were preserved for possible future evaluation. However, based on the results of the study, additional evaluations were not warranted, and the carcasses were discarded.
Postmortem examinations (parental animals):
POSTMORTEM EXAMINATIONS (PARENTAL ANIMALS)
SACRIFICE – Carbon dioxide asphyxiation, Random among all treatment groups within a sex
GROSS NECROPSY: All P1 adult rats, that were not found dead, were sacrificed by carbon dioxide anaesthesia and exsanguination. All adults received a gross pathological examination. The uteri of all cohabited females were examined for the presence and number of implantation sites in the uterus. Tissues were collected and preserved in appropriate fixative from all adult animals (See Table 1).
ORGAN WEIGHTS: Organ weights were recorded at necropsy (paired organs weighed together) for all P1 adult animals sacrificed by design. Group means and organ weight ratios (% body wt.) were calculated. Final body weight data were used for the calculation of organ to body weight ratios (% body wt.). (See Table 2).
HISTOPATHOLOGY: Microscopic examination was not conducted on any tissues from any P1 adult rats as it was not warranted based on reproduction data results.
Postmortem examinations (offspring):
POSTMORTEM EXAMINATIONS (OFFSPRING)
SACRIFICE
Pups prior to and on Lactation day 4 – Decapitation
Pups after Lactation day 4 and up to Lactation day 20 – Intraperitoneal injection of sodium pentobarbital
Pups age 21 days - Carbon dioxide asphyxiation, Random among all treatment groups within a sex
GROSS NECROPSY:
F1 Pups - F1 Pups (nursing offspring) that died (found dead, sacrificed in extremis, or accidentally killed) during the lactation period received a gross pathological evaluation. Tissues were not collected. Pups culled on post-natal-day (PND) 4 were discarded without a gross necropsy examination.
F1 Weanlings - All F1 weanlings were sacrificed by carbon dioxide anaesthesia and exsanguination. A gross pathological evaluation was performed on all weanlings. Gross lesions were preserved in formalin and the carcasses were discarded.
ORGAN WEIGHTS: F1 Pups and Weanlings - F1 pup and weanling organ weights were not recorded.
HISTOPATHOLOGY: Microscopic examination was not conducted on any tissues from any F1 pups or F1 weanlings.
Statistics:
See Table 3 for Statistical Analysis Methods
Reproductive indices:
REPRODUCTIVE INDICES
See Table 4 for Reproductive Function Indices for P1 Adult Rats
Offspring viability indices:
See Table 4 for Offspring Viability Indices for P1 Adult Rats

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
During Exposure - Clinical signs of convulsions were observed in animals during exposure to 3500 ppm. Two males exposed to 3500 ppm were found dead at the end of an exposure, which may be secondary to trauma sustained during a convulsive episode and one male was sacrificed in extremis, due to bilateral swelling of the hindlimbs, unrelated to exposure to the test substance. Unscheduled mortality did occur in 3 females during the lactation period. One female in the control group was sacrificed in extremis on lactation day 4 due to persistent vaginal bleeding, not test substance related since it occurred in the control group. One female in the 2000 ppm group was found dead on lactation day 5, not considered to be test substance related since mortality was not observed in 3500 ppm P1 females. One female in the 2000 ppm group was sacrificed in extremis on lactation day 0 due to dystocia, not considered test substance related since mortality was not observed in 3500 P1 females. There were no adverse, test substance-related clinical signs of toxicity observed during nonexposure periods for any group tested. All clinical signs observed were common to rats of this age, gender, strain, and pregnancy status.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Body Weight - P1 Male Rats
No adverse test substance-related effects on body weight or weight gain were observed in males for any exposure concentration. On test days 7 and 21, body weight of 3500 ppm males was significantly lower compared to the control mean, was similar to control values by the end of the exposure period, and was not considered biologically significant or adverse. Weight gain for 3500 ppm males was transiently decreased during test days 0 – 7, yet demonstrated recovery with significantly greater weight gain during test days 21 - 28. Weight gain was significantly greater in 2000 ppm males during test days 35 – 42, not test substance related.
Food Consumption – P1 Male Rats
On test days 0 – 7, food consumption of 3500 ppm males was significantly lower compared to the control mean, yet by the end of the exposure period, was similar to control values and not considered biologically significant or adverse. Food efficiency for 3500 ppm males was transiently decreased during test days 0 – 7; however, this group demonstrated recovery with significantly greater food consumption and body weight gain during test days 21 – 28. Food efficiency was significantly greater in 2000 ppm males during test days 21 – 28 and 35 – 42, not test substance related.
Food Consumption – P1 Female Rats During Premating
Food consumption was transiently, statistically greater in 500 and 3500 ppm females during test days 14 – 21. Since these statistical differences did not adversely impact body weight or weight gain, they were not considered to be biologically significant. Food efficiency was transiently statistically lower in 3500 ppm females during test days 63 – 70. Since this statistical difference did not adversely impact body weight or weight gain, it was not considered to be biologically significant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
The fertility index for 3500 ppm females was slightly lower than the control value (3500 ppm females had 83% fertility compared to control females which had 100% fertility). However, this value was within the historical control range of 72 – 100% for 30 studies conducted at the testing facility from 1998 to the time of study conduct. This slight difference was not considered test substance related. The number of implantation sites for 3500 ppm females was statistically greater compared to the control value with no biological relevance.

ORGAN WEIGHTS (PARENTAL ANIMALS):
P1 Female Rats
In P1 adult females exposed to 3500 ppm test substance, liver and pituitary gland weights were increased following 109 – 126 days of inhalation exposure to the test substance. Mean absolute and relative (% body wt.) liver weights were statistically significantly increased 14% and 11%, respectively, as compared to the control values. Mean absolute and relative (% body wt.) pituitary weights were statistically significantly increased 20% and 25%, respectively, as compared to the control values. (See Table 5).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
2 000 other: ppm (20618 mg/m3)
Sex:
male/female
Basis for effect level:
other: Clinical signs of toxicity observed during exposure to 3500 ppm and on the liver and pituitary weights (P1 females).
Dose descriptor:
NOAEL
Effect level:
3 500 other: ppm (36081 mg/m3)
Sex:
male/female
Basis for effect level:
other: highest concentration tested
Remarks on result:
other: Generation: reproduction (migrated information)
Dose descriptor:
NOAEL
Effect level:
3 500 other: ppm (36081 mg/m3)
Sex:
male/female
Basis for effect level:
other: highest concentration tested
Remarks on result:
other: Generation: offspring (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 5

Pathology

Mean Absolute and Relative (% body weight) Liver and Pituitary Weights

in P1Male and Female Rats

Sex:

Male

Female

Exposure (ppm):

0

500

2000

3500

0

500

2000

3500

 

 

 

 

 

 

 

 

 

Final Body Wt. (g.)

552.5

551.4

547.0

531.8

362.7

369.3

363.9

373.7

 

 

 

 

 

 

 

 

 

Liver (g.)

20.222

19.816

19.522

18.551

15.921

17.149

16.836

18.161*

% body wt.

3.660

3.573

3.563

3.488

4.380

4.643

4.616

4.873*

 

 

 

 

 

 

 

 

 

Pituitary (g.)

0.012

0.013

0.012

0.011

0.015

0.016

0.014

0.018**

% body wt.

0.002

0.002

0.002

0.002

0.004

0.004

0.004

0.005**

 

 

 

 

 

 

 

 

 

Underlined values were interpreted to be test substance-related weight effects.

* Statistically significant (Dunnett/Tamhane-Dunnett parametric comparison to control).

** Statistically significant (Dunn’s non-parametric comparison to control).

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity was 2000 ppm based on the clinical signs of central nervous system toxicity observed during exposure to 3500 ppm. The NOAEL for reproduction was 3500 ppm (36081 mg/m3).
Executive summary:

Twenty-five male and female rats per group were exposed 6 hours/day, 5 days or 7 days/week via repeated inhalation to atmospheric concentrations of 0, 500, 2000, or 3500 ppm test substance to evaluate the potential reproductive toxicity of the test substance. Concentrations of the test substance were generated by evaporation and subsequent dilution in conditioned, filtered air. Clinical observations, body weights, food consumption, and food efficiency were measured for P1rats. Offspring were evaluated for survival, clinical signs, and body weight on postnatal days 0, 4, 7, 14, and 21. At necropsy, P1 rats and offspring were given a gross evaluation at necropsy; selected organs from P1male and female rats were collected, weighed, and saved. No histopathological evaluation was warranted based on reproduction data results.

 

The mean atmospheric concentrations over the entire exposure period (± standard error of the mean) were 500 ± 2.1, 2000 ± 2.9, and 3500 ± 5.6 ppm in chambers targeted at 500, 2000, and 3500 ppm, respectively.

 

Mortality secondary to injuries sustained during convulsive episodes occurred in 2 of the P1 males exposed to 3500 ppm of test substance. Test substance-related clinical signs of convulsions were observed in P1 animals during exposure to 3500 ppm. There were no adverse, test substance-related effects on clinical observations evaluated during nonexposure periods or on body weight, weight gain food consumption or food efficiency in P1 males or females exposed to any concentration of the test substance. No adverse, test substance-related effects were observed on gross morphology, or any reproductive parameter evaluated in P1 males or females. Test substance-related increases in absolute and relative liver and pituitary weights occurred in 3500 P1 females. All organ weight parameters for P1 males were similar to the control values. There were no adverse, test substance-related effects on survival, body weights, clinical observations, or gross morphology in offspring produced from exposed P1 males or females. The No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity in P1 males and females was 2000 ppm based on the clinical signs of central nervous system toxicity observed during exposure to P1 males and females at 3500 ppm, and on the increased liver and pituitary weights in P1 emales. The NOAEL for reproduction and offspring was 3500 ppm (36081 mg/m3).