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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
fertility, other
Remarks:
Sub-chronic toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 413
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
fertility, other
Remarks:
Sub-chronic toxicity study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report equivalent or similar to OECD guideline 413
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 413
Principles of method if other than guideline:
Male/female rats were exposed to decalin vapor (0, 25, 50, 100, 200 or 400 ppm) for 6 hours plus 12 minutes/day, 5 days/week for 14 weeks. Reproductive tissue evaluations for male rats included epididymis, cauda epididymis and testis weights, spermatid measurements and epididymal sperm motility measurements. Female rats were assessed for all 4 stages of the estrous cycle.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic laboratory animals and services, Germantown, NY
- Diet (e.g. ad libitum except during exposure and urine collection periods): NTP-2000, Zeigler Bros., Inc, Gardners, PA
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 12 days (males), 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Relative humidity: 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Vapor was generated by pumping substance through a heated glass column filled with glass beads to increase surface area for evaporation. Heated nitrogen, pumped into the column, was used to vaporize the substance before transporting to exposure chamber at elevated temperature to avoid condensation. In exposure room, vapor was mixed with additional heated air in distribution manifold (at constant pressure) before being pumped through individual temeperature-controlled delivery lines to each exposure chamber. Vapor was diluted with conditioned chamber air to achieve desired exposure concentration.

Exposure chambers were designed to maintain a uniform vapor concentration throughout the chamber (total active mixing volume of 1.7 m3/chamber). Particle detector was used in exposure chambers to monitor particle count. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) ensuring that vapor and not aerosol was produced.

Build-up and decay rates for chamber vapor concentration were determined with animals present in chambers. At a chamber airflow rate of 15 air changes/ hour, time required to achieve 90% of target concentration (T90) was determined to be 12 minutes.
Details on mating procedure:
N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Substance concentration in exposure chambers were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes using a 12-port stream select valve. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of decalin in nitrogen supplied by a diffusion tube standard generator. The on-line gas chromatograph was calibrated monthly by a comparison of chamber concentration data to data from grab samples, which were collected with charcoal sampling tubes, extracted with toluene containing 1-phenylhexane as an internal standard, and analyzed by an off-line gas chromatograph. The volumes of gas were sampled at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of decalin containing 1-phenylhexane as an internal standard in toluene.
Duration of treatment / exposure:
6 hours + T90 (12 minutes)/day
Frequency of treatment:
5 days/week for 14 weeks
Details on study schedule:
Groups of 10 male and 10 female rats were distributed randomly into groups of approximately equal initial mean body weights (1 animal/cage). Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 6 weeks for clinical pathology analyses; additional groups of five male rats were exposed to the same concentrations for 2 weeks for renal toxicity analyses.
Remarks:
Doses / Concentrations:
0 (control), 25, 50, 100, 200 or 400 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, sham-exposed
Parental animals: Observations and examinations:
Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly.
Oestrous cyclicity (parental animals):
Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from core study females exposed to 0, 100, 200, or 400 ppm for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Sperm parameters (parental animals):
The following parameters were evaluated: spermatid heads per testis, per gram testis, per cauda, and per gram cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed.
Litter observations:
not examined
Postmortem examinations (parental animals):
Complete histopathology was performed on 0 and 400 ppm core study rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gall bladder (mice), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The kidneys from all male rats were also evaluated.
Postmortem examinations (offspring):
not examined
Statistics:
Organ and body weight data were analyzed by parametric multiple comparison procedures (Duunett, 1955; Williams, 1971 and 1972). Hematology, clinical chemistry, urinalysis, renal toxicity, and spermatid and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Reproductive indices:
not examined
Offspring viability indices:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
Increased liver and kidney weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased severity of hyaline droplets and granular casts in male rat kidneys
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-neoplastic – kidney lesions in male rats
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
No mortality was recorded in any of the animals. Mean body weight and body weight gains were similar to sham-exposed animals at study termination. Significant increases in glucose/creatinine, protein/creatinine, AST/creatinine and LDH/creatinine ratio was observed in male rats consistent with renal toxicity data. Mild increases were seen in AST/creatinine and LDH/creatinine ratio in female but did not correlate with the lack of kidney weight increases in females and hence not considered toxicologically relevant.
Transient increase in reticulocyte counts (day 3) and platelet counts (day 23) occurred in a dose-dependent trend in males and females. Changes were however minimal, had disappeared by study termination and were not considered toxicologically relevant.
Transient decreases in serum AP and increases in bile acid concentrations occurred on days 3 and 23 but had returned to control values by study termination. No changes were observed with serum ALT or SDH (other markers of hepatocellular injury) through the duration of the study.
Relative kidney weights and liver weights of male rats exposed to 50 ppm or greater and absolute kidney weights of 200 and 400 ppm males were significantly increased. Liver weights for 200 and 400 ppm females were also increased. Liver weight increases in the absence of pathological correlates were considered physiological adaptations to increased metabolic load.
Kidney lesions noted in males were associated with α2u-globulin nephropathy.

There were no significant differences between exposed and chamber control animals in sperm evaluations in males or vaginal cytology parameters in females.
Dose descriptor:
NOAEC
Effect level:
>= 400 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Highest dose tested
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Conclusions:
The NOAEC >=400 ppm for male/female for reproductive tissue evaluations and estrous cycle characterization
Executive summary:

Reproductive tissue for males and estrous cycles in females were evaluated following decalin (a C10 cycloparaffin) exposure. Groups of 25 male and 20 female F344/N rats were exposed to 0, 25, 50, 100, 200 or 400 ppm decalin vapor for 6 hrs/day, 5 days/week for 14 weeks. Samples were collected for sperm count, motility and vaginal cytology evaluations on rats exposed to 0, 100, 200 or 400 ppm. Parameters evaluated were spermatid counts per testis, per gram testis, per cauda and per gram cauda and epididymal spermatozoal motility. Left cauda, left epididymis and left testis were weighed. In females, vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from rats and mice for the purpose of vaginal cytology evaluations. Percentage of time spent in various estrous cycle stages and estrous cycle length were evaluated.

No clinical findings were noted to be related to decalin exposure. With respect to reproductive parameters, there was a statistically significant decrease in spermatid head counts (107)/testis in the 400 ppm dose group, however the significance of this finding does not appear to be clear or relevant to decalin exposure since there were no changes in spermatid counts /g testis, /g cauda epididymis or /cauda epididymis when compared with chamber controls. According to the NTP, “the decrease in spermatid heads/testis was not supported by other spermatid and sperm measurements and is considered of no biological significance”. Estrous cycle length of exposed females did not change compared to chamber controls.

Conclusion –Decalin exposure had no effect on sperm parameters of estrous cycle in male and female rats. NOAEC for fertility in both sexes ≥ 400 ppm (highest concentration tested).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 413
Principles of method if other than guideline:
Male/female rats were exposed to decalin vapor (0, 25, 50, 100, 200 or 400 ppm) for 6 hours plus 12 minutes/day, 5 days/week for 14 weeks. Reproductive tissue evaluations for male rats included epididymis, cauda epididymis and testis weights, spermatid measurements and epididymal sperm motility measurements. Female rats were assessed for all 4 stages of the estrous cycle.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Decalin (C10H18, decahydronaphthalene, CAS #91-17-8) was purchased from Sigma Aldrich Fluka (SAF) Bulk Chemicals (St. Louis, MO).The chemical, a colorless liquid, was identified as decalin by infrared and nuclear magnetic resonance spectroscopy and gas chromatography/mass spectrometry. The moisture content of lot 00334HR was determined by Karl Fischer titration; the purities of lots 13359 and 00334HR were determined by elemental analyses. Potentiometric titration was used to assess the purity of lots 07347LG, 12426EN, and 00334HR. The purities of all lots were also determined by gas chromatography. Elemental analyses for carbon and hydrogen were in agreement with the theoretical values for decalin. Karl Fischer titration indicated 73 ± 3.6 ppm water. Potentiometric titration detected no peroxides in lots 07347LG and 12426EN and 0.57 mEq/kg peroxides in lot 00334HR. Gas chromatography indicated two major peaks and up to seven impurities; the total area of the impurities did not exceed 0.59% of the total major peak areas. The overall purity was determined to be greater than 99%.
The bulk chemical was stored at room temperature, in metal drums under a nitrogen headspace. Stability was monitored using gas chromatography. No degradation of the bulk chemical was detected.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic laboratory animals and services, Germantown, NY
- Diet (e.g. ad libitum except during exposure and urine collection periods): NTP-2000, Zeigler Bros., Inc, Gardners, PA
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 12 days (males), 13 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 26
- Relative humidity: 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Vapor was generated by pumping substance through a heated glass column filled with glass beads to increase surface area for evaporation. Heated nitrogen, pumped into the column, was used to vaporize the substance before transporting to exposure chamber at elevated temperature to avoid condensation. In exposure room, vapor was mixed with additional heated air in distribution manifold (at constant pressure) before being pumped through individual temeperature-controlled delivery lines to each exposure chamber. Vapor was diluted with conditioned chamber air to achieve desired exposure concentration.

Exposure chambers were designed to maintain a uniform vapor concentration throughout the chamber (total active mixing volume of 1.7 m3/chamber). Particle detector was used in exposure chambers to monitor particle count. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) ensuring that vapor and not aerosol was produced.

Build-up and decay rates for chamber vapor concentration were determined with animals present in chambers. At a chamber airflow rate of 15 air changes/ hour, time required to achieve 90% of target concentration (T90) was determined to be 12 minutes.
Details on mating procedure:
N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Substance concentration in exposure chambers were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes using a 12-port stream select valve. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of decalin in nitrogen supplied by a diffusion tube standard generator. The on-line gas chromatograph was calibrated monthly by a comparison of chamber concentration data to data from grab samples, which were collected with charcoal sampling tubes, extracted with toluene containing 1-phenylhexane as an internal standard, and analyzed by an off-line gas chromatograph. The volumes of gas were sampled at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of decalin containing 1-phenylhexane as an internal standard in toluene.
Duration of treatment / exposure:
6 hours + T90 (12 minutes)/day
Frequency of treatment:
5 days/week for 14 weeks
Details on study schedule:
Groups of 10 male and 10 female rats were distributed randomly into groups of approximately equal initial mean body weights (1 animal/cage). Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 6 weeks for clinical pathology analyses; additional groups of five male rats were exposed to the same concentrations for 2 weeks for renal toxicity analyses.
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 25, 50, 100, 200 or 400 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, sham-exposed

Examinations

Parental animals: Observations and examinations:
Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly.
Oestrous cyclicity (parental animals):
Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from core study females exposed to 0, 100, 200, or 400 ppm for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Sperm parameters (parental animals):
The following parameters were evaluated: spermatid heads per testis, per gram testis, per cauda, and per gram cauda and epididymal spermatozoal motility. The left cauda, left epididymis, and left testis were weighed.
Litter observations:
not examined
Postmortem examinations (parental animals):
Complete histopathology was performed on 0 and 400 ppm core study rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gall bladder (mice), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (except male mice), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The kidneys from all male rats were also evaluated.
Postmortem examinations (offspring):
not examined
Statistics:
Organ and body weight data were analyzed by parametric multiple comparison procedures (Duunett, 1955; Williams, 1971 and 1972). Hematology, clinical chemistry, urinalysis, renal toxicity, and spermatid and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1951) were examined and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.
Reproductive indices:
not examined
Offspring viability indices:
not examined

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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:
effects observed, treatment-related
Description (incidence and severity):
Increased liver and kidney weights
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased severity of hyaline droplets and granular casts in male rat kidneys
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
non-neoplastic – kidney lesions in male rats
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

No mortality was recorded in any of the animals. Mean body weight and body weight gains were similar to sham-exposed animals at study termination. Significant increases in glucose/creatinine, protein/creatinine, AST/creatinine and LDH/creatinine ratio was observed in male rats consistent with renal toxicity data. Mild increases were seen in AST/creatinine and LDH/creatinine ratio in female but did not correlate with the lack of kidney weight increases in females and hence not considered toxicologically relevant.
Transient increase in reticulocyte counts (day 3) and platelet counts (day 23) occurred in a dose-dependent trend in males and females. Changes were however minimal, had disappeared by study termination and were not considered toxicologically relevant.
Transient decreases in serum AP and increases in bile acid concentrations occurred on days 3 and 23 but had returned to control values by study termination. No changes were observed with serum ALT or SDH (other markers of hepatocellular injury) through the duration of the study.
Relative kidney weights and liver weights of male rats exposed to 50 ppm or greater and absolute kidney weights of 200 and 400 ppm males were significantly increased. Liver weights for 200 and 400 ppm females were also increased. Liver weight increases in the absence of pathological correlates were considered physiological adaptations to increased metabolic load.
Kidney lesions noted in males were associated with α2u-globulin nephropathy.

There were no significant differences between exposed and chamber control animals in sperm evaluations in males or vaginal cytology parameters in females.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
>= 400 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEC >=400 ppm for male/female for reproductive tissue evaluations and estrous cycle characterization
Executive summary:

Reproductive tissue for males and estrous cycles in females were evaluated following decalin (a C10 cycloparaffin) exposure. Groups of 25 male and 20 female F344/N rats were exposed to 0, 25, 50, 100, 200 or 400 ppm decalin vapor for 6 hrs/day, 5 days/week for 14 weeks. Samples were collected for sperm count, motility and vaginal cytology evaluations on rats exposed to 0, 100, 200 or 400 ppm. Parameters evaluated were spermatid counts per testis, per gram testis, per cauda and per gram cauda and epididymal spermatozoal motility. Left cauda, left epididymis and left testis were weighed. In females, vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from rats and mice for the purpose of vaginal cytology evaluations. Percentage of time spent in various estrous cycle stages and estrous cycle length were evaluated.

No clinical findings were noted to be related to decalin exposure. With respect to reproductive parameters, there was a statistically significant decrease in spermatid head counts (107)/testis in the 400 ppm dose group, however the significance of this finding does not appear to be clear or relevant to decalin exposure since there were no changes in spermatid counts /g testis, /g cauda epididymis or /cauda epididymis when compared with chamber controls. According to the NTP, “the decrease in spermatid heads/testis was not supported by other spermatid and sperm measurements and is considered of no biological significance”. Estrous cycle length of exposed females did not change compared to chamber controls.

Conclusion –Decalin exposure had no effect on sperm parameters of estrous cycle in male and female rats. NOAEC for fertility in both sexes ≥ 400 ppm (highest concentration tested).