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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09.12.2003 to 20.06.2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006
Reference Type:
other: Study report amendment
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 7 weeks
- Weight at study initiation: Males (P): 243 - 313 g; Females: 141 - 196 g; Males (F1): 381 - 603 g; Females: 223 - 362 g
- Fasting period before study: None
- Housing: Individually in stainless steel wire-mesh cages, mating in home cage of male, following mating females were removed to plastic maternity cages until lactation day 21, then transferred back to wie-mesh cages.
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3
- Humidity (%): 50± 20
- Air changes (per hr): Minimum 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 03.12.2003 To: 12.12.2005

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.0 m3 stainless steel and glass whole body inhalation chambers
- Method of holding animals in test chamber: None
- Source and rate of air: No data
- Method of conditioning air: No data
- Temperature, humidity, pressure in air chamber: 19-27oC, 34-66%, slight negative pressure, respectively
- Air flow rate: No data
- Air change rate: 12-15 changes/hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes, samples were taken from the approximate middle of each chamber
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days (or until evidence of mating)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): in a plastic maternity cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Duration of treatment / exposure:
At least 70 days prior to mating, throughout mating, gestation through gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia . Premating exposure period (males): 70 days.
Premating exposure period (females): 70 days. Duration of test: appproximately 18 months.
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
- F1 parental animals not mated until approximately 11 weeks (not stated) after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 3 weeks of age.
- Age at mating of the mated animals in the study: 14 -16 weeks.
Doses / concentrationsopen allclose all
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
1 600 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the results of a previous study (no details given)
- Rationale for animal assignment (if not random): random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for appearance, behavior moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly body weights and food consumption were recorded on gestation days (GD) 0, 4, 7, 11, 14 and 20 and on Postnatal days (PND) 1, 4, 7, 14 and 21 for females in the F0 and F1 generations.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to pairing. Ovarian primordial follicle counts were recorded for all F1 females in the control and high exposure groups. 
Sperm parameters (parental animals):
Spermatogenic endpoints (sperm motility [including progressive motility], morphology and numbers) were recorded for all F0 and F1 males. 
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Each litter was examined to determine the number of fetuses, sex, still births runts, survival, litter viability and death. 

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Forty pups/sex/group from the F2 generation were selected for evaluation of surface-righting (PND 5 to day of positive response) and air-righting (PND 13, 17, 21 and 61) reflexes; of these, 20 F2 pups/sex/group were selected for FOB (PND 4, 11, 21, 45 and 60), auditory startle response (PND 20 and 60), locomotor activity (PND 13, 17, 21 and 61) and/or learning and memory assessment (PND 22 or 62). 
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS

Organs examined at necropsy (macroscopic and microscopic): Complete detailed necropsy was conducted. Organ weights: Adrenal glands, brain, epididymis (total and cauda), kidneys, liver, lungs, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands and accessory fluids, spleen, testes, thyroid gland, uterus with oviducts and cervix   Histopathologic evaluation: Adrenal glands, brain, cervix, coagulated glands, epididymis (right), kidneys, liver, lungs, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, thyroid gland, uterus, vagina, vas deferens, all gross (internal) lesions. 
Postmortem examinations (offspring):
Nonselected F1 pups were necropsied on PND 21 or 28, and nonselected F2 pups were necropsied on PND 21.  Selected organs were weighed from F1 and F2 pups (one/sex/litter) that were necropsied on PND 21.  Selected F2 rats not allocated for neuropathology and brain dimension measurements were necropsied following completion of reflex ontogeny evaluations (PND 61) or at study termination (PND 72).  Each surviving  F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F2 pups; selected organs were weighed.  

Organs examined at necropsy (macroscopic and microscopic): Complete detailed necropsy was conducted. Organ weights: Adrenal glands, brain, epididymis (total and cauda), kidneys, liver, lungs, ovaries, pituitary gland, prostate gland, seminal vesicles with coagulating glands and accessory fluids, spleen, testes, thyroid gland, uterus with oviducts and cervix   Histopathologic evaluation: Adrenal glands, brain, cervix, coagulated glands, epididymis (right), kidneys, liver, lungs, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, testes, thyroid gland, uterus, vagina, vas deferens, all gross (internal) lesions. 
Statistics:
Statistical methods: Parametric analysis was screened for homogeneity of variance using Levene's test and normality using Shapiro-Wilk's test. If the data was not homogenous and normal, then the data were analyzed using nonparametric statistics (Kruskal-Wallis ANOVA test followed by the Mann-Whitney U-test). Homogeneous data was analyzed by Chi-Square test with Yates correction factor, One-way ANOVA with Dunnett's test and Kolmogorov-Smirnov test (one-tailed test).  FOB data and histopathological findings were compared to the control group using a two-tailed Fisher's Exact test.  P< 0.05 or P < 0.01.
Reproductive indices:
Reproductive parameters (days between pairing and coitus, length of gestation, evidence of parturation difficulties, mating, fertility, copulation and conception indices, testicular and epididymal sperm counts, sperm production rate sperm motility and morphology, ovarian primordial follicle count), developmental landmarks (e.g., balanopreputial separation and vaginal patency).
Offspring viability indices:
Mean litter size, postnatal survival (postnatal day 0 to 4), postnatal survival for other intervals up to day 21.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No test article-related mortalities or clinical findings were observed in this study. One female in the F1 generation (highest dose group) died of dystocia (17 dead fetused in utero).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Lower weekly body weight gains were noted for F0 males and females in the 1600 and 5000 ppm groups and F1 males in the 5000 ppm group.  Mean body weights in the 1600 ppm group for both the F0 and F1 generations were generally similar to control group values, while those in the 5000 ppm group were reduced throughout the majority of both the F0 and F1 generations. Food consumption was lower for the 5000 ppm group males during the premating period (F0) and throughout the entire generation (F1).  Food consumption for F1 females in the 5000 ppm group was reduced during the first week following weaning (week 17-18) only. 

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): No adverse effects.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): No adverse effects.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No adverse effects.

ORGAN WEIGHTS (PARENTAL ANIMALS): Test article-related higher kidney weights were noted for F0 and F1 males in the 1600 and 5000 ppm groups.  Corresponding histopathological effects of HMDS in this study were similar to those previously reported in rats. Higher mean relative liver weights were noted for the F1 males in 1600 and 5000 ppm groups.

GROSS PATHOLOGY (PARENTAL ANIMALS): No adverse treatment-related findings.

HISTOPATHOLOGY (PARENTAL ANIMALS): Test article-related higher kidney weights were noted for F0 and F1 males in the 1600 and 5000 ppm groups.  Corresponding histopathological effects of HMDS in this study were similar to those previously reported (Cassidy et al., 2001) in rats following long-term inhalation exposure at 593 and 5012 ppm.  These findings included hyaline droplets (F0 and F1 males at 5000 ppm) and increased incidence and severity of basophilic tubules in the kidneys (F0 males, F1 males and F1 females at 5000 ppm).  Male rat-specific hyaline droplet (consistent with alpha 2 urinary globulin) nephropathy was associated with the increase in basophilic tubules.  Other test article related microscopic findings, including golden-brown pigment in the periportal areas of the liver for F0 males in the 5000 ppm group and F1 males and females in the 1600 and 5000 ppm groups.  This pigment was accompanied by infiltration of primarily mononuclear inflammatory cells and/or bile duct hyperplasia in the liver in the F0 and F1 5000 ppm groups, and corresponded to higher mean relative liver weights for the F1 males in the 1600 and 5000 ppm groups.  The golden-brown pigment demonstrated bright red birefringence with a central dark Maltese cross under polarized light, characteristic of porphyrin (Churukian, 2002).  In the 5000 ppm group F1 males and females, golden brown pigment was also noted in the medullary macrophages of the mesenteric lymph nodes and alveolar macrophage aggregates were noted for F0 and F1 males and females in the 5000 ppm group. Effects in the lungs were diagnosed as idiopathic rat respiratory syndrome, and were not therefore related to treatment.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicity: Based on effects in the liver.
Dose descriptor:
NOAEC
Effect level:
>= 5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity: No reproductive toxicity in the parent animals of the P and F1 animals.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): There were no treatment-related effects in postnatal survival of the F1 litter.


CLINICAL SIGNS (OFFSPRING): None


BODY WEIGHT (OFFSPRING): Offspring body weight gains were decreased for F1 pups in the 5000 ppm group from PND 4-21 and F2 pups in the 1600 and 5000 ppm groups from PND 4 to 14.  In the 5000 ppm group, these reduced body weight gains resulted in substantially lower mean body weights on PND 14 and 21 in both generations (approximately 6% to 7% lower by PND 21); lower body weights continued to be observed for the F2 males and females through PND 56 and PND 35, respectively.  In the F2 pups in the 1600 ppm group, the lower body weight gain during PND 4-14 resulted in significantly lower mean body weights on PND 14 (approximately 7% and 5% for male and female pups, respectively); lower body weights were also observed for the F2 males and females in this group through PND 49 and 35, respectively.


SEXUAL MATURATION (OFFSPRING): Balanopreputial separation and vaginal patency were not affected in the F1 pups.


ORGAN WEIGHTS (OFFSPRING): On PND 21, decreased thymus weights were noted for F2 male pups in the 1600 and 5000 ppm groups and F2 female pups in the 5000 ppm group only. There were no such findings in the F1 pups.


GROSS PATHOLOGY (OFFSPRING): There were no remarkable findings in the F1 and F2 pups that were found dead or euthanised in extremis. There were no abnormal findings in the F1 and F2 pups.


HISTOPATHOLOGY (OFFSPRING): No treatment-related effects.


OTHER FINDINGS (OFFSPRING): F1 adult females and their offspring exhibited no exposure-related effects during the FOB assessments. No test article related effects on air-righting reflex or learning and memory were noted for F2 offspring. The only test substance-related effect on attainment of developmental landmarks noted for F2 pups was a slight delay in attainment of the surface righting response for females in the 5000 ppm group (65% attained the response on PND 5 versus 90% in the control group).

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicity: Based on effects in the liver.

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEC
Generation:
F2
Effect level:
1 600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on decreased body weight gains in the F2 generation animals at 5000 ppm.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

There were no statistically significant offspring weights at any dose level. Statistically significant (p<0.05 or p<0.01) lower mean pup body weight gains were noted for males in the 400, 1600 and 5000 ppm groups and for females in the 400 and 5000 ppm groups from PND 4 to PND 7. Male and female pup weight gain in the 5000 ppm group continued to be slightly lower (not statistically significant) than the control group during PND 7 to 14 and 14 to 21. As a result of the lower weight gain, mean pup body weights in the 5000 ppm group were lower (7.2% to 7.3%) than control group values on PND 21; the difference from the control group for the females was statistically significant (p<0.05). Because the lower weight gain in the 400 and 1600 ppm groups did not persist, the lower mean body weight gains in these groups during PND 4 to 7 were not considered test article-related. No other differences in pup body weight or body weight gain relative to the control group were noted in the 400, 1600 or 5000 ppm groups. Mean male and female pup body weights and body weight changes in the 100 ppm group were unaffected by test article exposure throughout the postnatal period. The NOAEC for F1 developmental toxicity was 1600 ppm due to decreased offspring weights at 5000 ppm. There were no gross anomalies in the pups. The NOAEC for developmental neurobehavioral endpoints was 1600 ppm due to the lack of habituation exhibited in the locomotor activity assessments and delayed attainment of the surface righting response in the 5000 ppm group F2 females, and decreases in average and peak acoustic startle response on PND 20 in the 5000 ppm group F2 males and females. The NOAEC for neuropathologic endpoints was 5000 ppm.

Table 1 - SUMMARY OF OFFSPRING WEIGHTS (F1- pre-weaning) [G] (LITTER AS EXPERIMENTAL UNIT)      

GROUP:                

0 PPM           

100 PPM           

400 PPM          

1600 PPM          

5000 PPM 

PND 1

MALES               

MEAN                 

7.0               

6.9         

7.1 

7.0 

6.9

S.D.  

0.59

0.54

0.90

0.49

0.70

N 

27              

29

26

27                 

25

FEMALES               

MEAN                   

6.6

6.5

6.7

6.6

6.6

S.D.                  

0.60

0.58

0.78

0.47

0.67

N

27

29

26

27

25

PND 4 (BEFORE SELECTION)

MALES

MEAN                   

10.0

9.9

10.5

9.8

10.2

S.D.                  

1.08

1.00

1.44

0.99

1.12

N 

27

29

26

27

25

FEMALES

MEAN                   

9.6

9.4

9.8

9.3

9.7

S.D.                  

1.06

1.0.4

1.32

0.98

1.23

N 

27

29

26

27

25

PND 7

MALES

MEAN                   

14.3

14.0

14.3

13.7

13.6

S.D.                  

1.63

1.79

1.82

1.59

1.25

N 

27

28

26

27

25

FEMALES

MEAN                   

13.7

13.2

13.4

13.0

13.1

S.D.                  

1.57

1.80

1.78

1.56

1.31

N 

27

28

26

27

25

PND 14

MALES

MEAN                   

25.8

26.0

26.1

24.9

24.4

S.D.                  

2.33

2.18

2.46

2.88

2.09

N 

27

27

25

27

25

FEMALES

MEAN                   

24.9               

25.0               

24.8               

24.0               

23.6

S.D.                  

2.28               

1.93               

2.43               

2.56               

2.27

N 

27                                                                      

27

25

27

25    

PND 21

MALES

MEAN                   

41.3               

42.0               

40.9               

39.5               

38.3               

S.D.                  

4.42               

4.19               

5.09               

4.22               

4.13

N 

27                                                                                

27

25

27

25

FEMALES               

MEAN                   

40.1               

40.2               

38.8               

37.9               

37.2*               

S.D.                  

4.30               

3.91               

4.62               

3.99               

4.06

N 

27                                                                     

27

25  

27 

25

PND = POSTNATAL DAY 

MODIFIED STATISTICS USED. 

* = Significantly different from the control group at 0.05

 

  Table 2 - SUMMARY OF OFFSPRING WEIGHT CHANGES [G] (F1- pre-weaning) (LITTER AS EXPERIMENTAL UNIT)

GROUP:                

0 PPM           

100 PPM           

400 PPM          

1600 PPM          

5000 PPM 

PND 1-4 (BEFORE SELECTION)

MALES               

MEAN                 

3.0                

3.0                

3.3                

2.8                

3.2

S.D.  

0.62               

0.67               

0.75               

0.64               

0.74

N 

27                                                                             

29 

26

27

25 

FEMALES               

MEAN                   

2.9                

2.8                

3.1                

2.7                

3.1               

S.D.                  

0.62               

0.68               

0.75               

0.64               

0.79                

N

27                                                                      

29

26

27

25

PND 4 TO 7

MALES

MEAN                   

4.4                

4.1                

3.8+               

3.9+               

3.5++

S.D.                  

0.68               

1.09               

0.97               

0.95               

0.71

N 

27                                                                            

28

26 

27  

25

FEMALES

MEAN                   

4.2                

3.8                

3.6+               

3.7                

3.4++

S.D.                  

0.67               

1.01               

0.89               

0.93               

0.67                

N 

27                                                                            

28

26

27

25

PND 7 TO 14

MALES

MEAN                   

11.4               

11.9               

11.9               

11.2               

10.8

S.D.                  

1.42               

1.44               

1.28               

1.83               

1.11

N 

27                                                                   

27

25

27   

25

FEMALES

MEAN                   

11.2               

11.6               

11. 4               

11.0               

10.5

S.D.                  

1.41               

1.20               

1.22               

1.64               

1.23                

N 

27                                                 

27

25   

27                 

25    

PND 14 TO 21

MALES

MEAN                   

15.5               

16.0               

14.8               

14.6               

13.9

S.D.                  

2.61               

2.74               

3.17               

2.06               

2.48

N 

27                 

27                                                  

25

27  

25

FEMALES

MEAN                   

15.2               

15.2               

14.1               

14.0               

13.6               

S.D.                  

2.41               

2.66               

2.85               

2.28               

2.28                

N 

27                                                                           

27

25

27

25

PND = POSTNATAL DAY MODIFIED STATISTICS USED. 

* INDICATES PARAMETRIC ANALYSIS AND + INDICATES NON-PARAMETRIC ANALYSIS. 

Applicant's summary and conclusion

Conclusions:
In a two-generation reproductive toxicity study on HMDS, conducted to GLP (reliability score 1) the NOAEC for parental toxicity relevant to humans was 400 ppm based on microscopic liver findings in the F0 males of the 5000 ppm group and F1 males and females in the 5000 and 1600 ppm groups. F0 and F1 reproductive performance was not affected at any concentrations. In the F2 generation, offspring body weight gains were persistently decreased in the 5000 ppm groups from PND 4-14, and this lead to decreased body weights at late as PND 49 in male offspring. The few “findings” at the 1600 ppm and lower exposure levels lacked consistency (along the time line), were of insufficient magnitude, and insufficiently dose-responsive. F1 adult females and their offspring exhibited no exposure-related effects during the FOB assessment. Based on the results of this study the NOAEC for HMDS for parental reproductive toxicity was considered to be at least 5000 ppm. The NOAEC for neonatal toxicity was considered to be 1600 ppm due to decreased offspring weights at 5000 ppm.