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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to OECD Testing Guideline 416 (Two-Generation Reproduction Toxicity Study). GLP.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Assessment in rats of the reproductive toxicity of gasoline from a gasoline vapor recovery unit
Author:
McKee, R. H., G. W. Trimmer, F. T. Whitman, C. S. Nessel, C. R. Mackerer, R. Hagemann, R. A. J. Priston, A. J. Riley, G. Cruzan, B. J. Simpson, J. H. Urbanus
Year:
2000
Bibliographic source:
Reproductive Toxicology. 2000. 14:337-353.
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: low viscosity liquid hydrocarbon
Details on test material:
The test material used was vapor recovery units (VRU) gasoline collected during 1995 from a distribution terminal in the Netherlands. The test material was identified in the European Inventory of Existing Chemical Substances (EINECS) as vapor-recovery gasoline, CAS No. 68514-15-8. The vapor recovery unit in use at that terminal used Kaldair Technology. The VRU recovers hydrocarbons from the inlet stream by separating the volatile organic constituents from air in a pair of carbon adsorption beds, and to obtain condensate, a desorption step is necessary. The constituents are then condensed to form a recovered product. Thus, based on the characteristics of the VRU, it seemed likely that the recovered material would effectively be the volatile fraction of gasoline to which exposure occurs. To verify the representative nature of the test material, samples of vapor from the terminal and condensed product were taken for chemical analysis. These analyses were then compared with vapor measurements at another terminal and to the results of gasoline exposure studies, particularly those relating to customers at self-service stations. These analyses were all in reasonable agreement and a large volume of VRU condensate was collected for toxicological testing.

See attachment for non aromatics, toluene and benzene.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The experimental and control rats were placed (whole body) into appropriate inhalation chambers which were operated under dynamic conditions. The rats were exposed six hours per day, seven days/week, and remained in the chambers as the test atmosphere cleared for a minimum of the theoretical equilibration time (T99 = 23 minutes). The test material was administered fully vaporized in the breathing air of the animals. The test atmosphere composition and concentration remained constant at each exposure level over the daily six-hour period. All P1 and P2 adult male rats were exposed for at least 10 weeks prior to mating, through the mating period for F1 and F2 litters and until their sacrifice. All P1 and P2 adult female rats were exposed for at least 10 weeks prior to mating, during the mating period, and through Gestation Day 20. Exposure was resumed on postpartum Day 5, and continued until the female rats were sacrificed. Female rats that did not deliver or were not confirmed mated were exposed until their sacrifice.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall mean chamber concentrations of VRU gasoline during the exposure periods throughout the study were 0 +/- 0, 5076 +/- 146, 10247 +/- 249, and 20241 +/- 373 mg/m3. The concentrations of the test atmosphere were determined approximately hourly during each exposure by on-line gas chromatography (Hewlett Packard GC 6890).
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
7 days/ week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5000, 10000, 20000 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
30 males and 30 females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: The highest concentration was chosen to correspond to approximately half of the lower explosive limit, considered the highest level safe to test.
Positive control:
None tested.

Examinations

Parental animals: Observations and examinations:
All test animals were checked twice daily for viability, and clinical observations were carried out on a daily basis. Body weights and food consumption were measured weekly until confirmation of mating and then on GD 0, 7, 14, and 21 and PPD 0, 4, 7, 14, and 21 for P1 and P2, and on PPD 28 for P1 only.
Oestrous cyclicity (parental animals):
There was also an ovarian examination that included confirmation of growing follicles and corpora lutea and quantification of primordial oocytes. Five sections per ovary for all ovaries from the high-dose and control group animals were evaluated under light microscopy. As there were no differences between the high dose (20000 mg/m3) and control, tissues from animals in the intermediate groups were not examined. Reproductive parameters evaluated included: male and female fertility indices, male mating index, female fecundity and gestational indices, mean litter size, mean days of gestation, female estrous cycle length, and number of females cycling normally.
Sperm parameters (parental animals):
Samples of sperm from the left distal cauda epididymis were collected from all males at terminal sacrifice for evaluation of sperm parameters. These included assessments of total caudal epididymal sperm number, percent progressively motile sperm, and homogenization resistant spermatid count, percent morphologically normal sperm, and percent of sperm with an identified abnormality. To assess progressively motile sperm, the left cauda was sliced and suspended in a petri dish in 10 ml Dulbecco’s phosphate- buffered saline (PBS) with 1.0% bovine serum albumen at 37°C for 15 min. The resulting suspension was swirled gently, and a 1.0-ml sample was added to 9 ml warmed media. A sample was taken up in a cannula by capillary action and assessed by the Hamilton Thorne Research IVOS (Integrated Visual Optical System) from the suspension in the petri dish. Total cauda epididymal sperm counts were also assessed by the IVOS. For evaluation of homogenization-resistant spermatid counts, the testes were placed in 20—30 ml SMT solution (100 mg merthiolate and 0.5 ml Triton X-100 in 1000 ml 0.9% saline), homogenized for 2 min, and allowed to settle. The volume was brought up to 50 ml SMT solution, homogenized again, and allowed to settle for 1 min. An aliquot of the homogenate was then stained for DNA, and quantified using the IVOS system.

Sperm morphology was determined manually under phase contrast microscopy. Ten males were randomly selected from each treatment group. Samples were collected from the left cauda of each animal, and four slides were prepared from each sample. Two of the slides were stained with 1.0% Eosin Y and two with Papanicolaou stain. The Eosin Y slides were evaluated, and the Papanicolaou stained slides were retained for future evaluation. Five hundred sperm from each animal were evaluated.
Litter observations:
All pups were counted and examined externally on a daily basis until PND 21, and weighed on PND 0, 4, 7, 14, and 21. F1 pups were also examined daily from PND 21 to 28 and weighed on PND 28 and 35. All surviving F1 and F2 pups were evaluated for developmental landmarks, including pinna detachment, hair growth, incisor eruption, eye opening, and the development of the surface righting reflex.
Postmortem examinations (parental animals):
For the adult animals, organs weighed included liver, adrenals, brain, uterus, testes, right epididymis, and left caudal epididymis, seminal vesicles (with coagulating glands and fluid), kidneys, spleen, thymus, ovaries, prostate, and lungs. The list of tissues taken for microscopic examination included vagina, uterus, ovaries, right epididymis, seminal vesicles, prostate, oviducts, thymus, trachea, nasal turbinates, spleen, coagulating gland, pituitary, kidneys, liver, mammary gland (females only), testes, brain, tissue masses/gross lesions, larynx, lungs, and adrenals. The tissues from the high-dose (20000 mg/m3) and control animals were evaluated. Kidney sections from all exposure groups were examined.
Postmortem examinations (offspring):
All animals dying spontaneously or sacrificed in a moribund condition were necropsied. Culled pups were examined externally but were not necropsied unless there was external evidence of abnormalities. Randomly selected pups were necropsied, and the following organs were weighed: ovaries, liver, adrenals, thymus, testes, kidneys, spleen, and brain. Additionally, the following tissues were taken for microscopic examination: vagina, ovaries, epididymides, prostate, pituitary, spleen, kidneys, thymus, uterus (with cervix), testes, seminal vesicles, coagulating gland, adrenals, liver, brain, and any gross lesions. The majority of the tissues were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, and stained with hematoxylin and eosin (H&E). The right testes were fixed in Bouin’s fixative and stained with the periodic acid-Schiff reaction (PAS). Sections of the kidneys of male rats from all groups of the P1 and P2 generations were stained by Mallory’s Heidenhain technique for the identification of hyaline droplet accumulation.
Statistics:
See "Other Information" for additional details.

Continuous data were first evaluated by Bartlett’s test of homogeneity of variance to determine whether the groups had equivalent variances at the 1% level of significance. If the variances were equivalent, the hypothesis that there was no difference in response between groups was tested by standard one-way analysis of variance (ANOVA). If the ANOVA was significant, Dunnett’s test was used to determine which treated groups differed from control. A linear regression to test for a dose-response was also carried out and tested for lack of fit. If the variances were not equivalent, a Kruskal-Wallis (non-parametric) test was used to determine whether the effects were equivalent. If there was a difference, Dunn’s Rank Sum comparison was used to determine which groups differed from control. Jonckheere’s test for ordered response was also performed.
Reproductive indices:
Reproductive parameters evaluated included: male and female fertility indices, male mating index, female fecundity and gestational indices, mean litter size, mean days of gestation, female estrous cycle length, and number of females cycling normally. Live birth index, survival indices (PPD 1, 4, 7, 14, 21), viability index at weaning, mean live and dead offspring on Day 0, sex ratio at Day 0, offspring inlife observations, offspring body weight, and offspring gross postmortem findings were also assessed.
Offspring viability indices:
All pups were counted and examined externally on a daily basis until PND 21, and weighed on PND 0, 4, 7, 14, and 21. F1 pups were also examined daily from PND 21 to 28 and weighed on PND 28 and 35. All surviving F1 and F2 pups were evaluated for developmental landmarks, including pinna detachment, hair growth, incisor eruption, eye opening, and the development of the surface righting reflex. All surviving F1 female offspring were monitored for vaginal opening beginning on PND 29, and F1 male offspring were monitored for preputial separation beginning on PND 35.

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
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:
no effects observed

Details on results (P0)

There were no treatment-related clinical signs of toxicity or mortality. In the P1 generation, there were two deaths during the exposure period, one control male and one female in the 5000 mg/m3 group. In the second parental generation, one male in the control group and one in the 10 000 mg/m3 group died during exposure. The deaths were not considered treatment related. All animals in the highest exposure group survived in both generations. Analysis of the data revealed that there were no significant effects on weight gain or food consumption between treated and control animals of either generation. Similarly, there were no differences in gestational or postpartum body weights or food consumption in either generation. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related.

There were no significant differences in absolute organ weights in either males or females from the P1 generation. Among the second parental (P2) generation animals, there were some statistically significant increases in absolute organ weights, including liver, kidney, and testis in the males and lungs in the females, but none of the organ weight differences between the high exposure group and control animals were significantly different. In the absence of clear dose-response relationships, the toxicological significance of these data is unclear. When the data were expressed on an organ to body weight basis, the only significant differences were an elevation of relative kidney weights in the males from the low exposure group of the first parental generation (in the absence of a dose-response this observation was assumed not to be treatment-related) and an elevation of relative kidney weights from the high-exposure group males from the second parental generation. The latter observation may have been treatment-related, but, as described below, was not clinically important.

HISTOPATHOLOGY

There were no compound-related microscopic changes in any of the reproductive tissues or in the tissues of the upper or lower respiratory tract from any of the P1 or P2 generation rats exposed to 20000 mg/m3 VRU gasoline. The only treatment-related histologic changes were observed in the kidneys of male rats of both generations and consisted of exposure-related increases in the amount and size of hyaline droplets. The hyaline droplets in several of the exposed rats were larger and stained more densely with the Heidenhain stain. The enlargement of these droplets was due to coalescing of clusters of hyaline droplets and the accumulation of irregular to somewhat angular-shaped hyaline droplets. The only other treatment related effect in the kidney was seen in three male rats of the high-dosage groups from both the P1 and P2 generations and consisted of granular casts in medullary tubules. These granular casts often accompany increased hyaline droplet accumulations and are consistent with the “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of alpha-2-u-globulin in the kidney. This finding is not relevant to human risk assessment.

Reproductive parameters

In the first parental generation, there were no differences in mating index, fecundity, pregnancy, or length of gestation. The results in the second parental generation were similar.

Sperm parameters and Estrous cycle

The sperm analysis was carried out on both P and on P2(F1) males. There were no significant effects on sperm count, progressive motility, or gross appearance in either group. There were no statistically significant differences in mean estrous cycle length, quantification of primordial oocytes, or percent females with abnormal cycles between treated and control females in the P1 or P2 generations.

Effect levels (P0)

Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
>= 20 000 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive parameters.

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Pathological Examinations

As noted for the parental animals, the only treatment-related histologic changes were observed in the kidneys of F1 male rats consisted of exposure-related increases in the amount and size of hyaline droplets, granular casts in medullary tubules of the kidney. These findings are consistent with “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of alpha-2-u-globulin in the kidney. This finding is not relevant to human risk assessment.

Reproductive parameters

Among the offspring of the first parental generations, there were no differences in mean litter size, fraction of live births, or sex ratio. The results in the second parental generation were similar. Among the offspring, there were no differences in survival of offspring through weaning in the first generation, and, in the second generation, early survival was slightly higher among the offspring from exposed dams than those from controls. There were no differences in the weight of the offspring through weaning in either generation. Finally, there were no unusual postmortem observations that were considered to be treatment related.

Developmental landmarks

There were no significant differences in incisor eruption, pinna detachment, or surface righting reflex in the F1 or F2 offspring, or vaginal patency or preputial separation in the F1 offspring. There was a significant delay in hair growth in the males but not the females of the F1 pups. Eye opening was advanced by approximately one-half a day for the high-dose males, and hair growth was delayed in the low-dose females of the F2 offspring.

Effect levels (F1)

Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
>= 20 000 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive parameters.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the data reported, the reproductive NOAEL as defined by this study is >20000 mg/m3, the highest dose tested and approximately half of the lower explosive limit.
Executive summary:

The generational reproductive study was conducted at levels up to 20000 mg/m3, approximately half of the lower explosive limit, and the highest level considered safe for use in the laboratory. VRU gasoline did not produce any pathologic changes in reproductive organs. Additionally, there were no differences in mating, fertility, live births, birth weights, and survival or weight gain through weaning. Finally, there were no differences in sperm count, sperm quality, estrous cycling, quantification of primordial oocytes, or developmental landmarks, other than a delay in hair growth in some treated offspring.

 

There were weight and histopathological changes noted in the kidneys of the high-dose (20000 mg/m3) exposed males from the second parental generation, as well as microscopic evidence of hyaline droplets in the male rat kidneys from both generations. However, as the weight difference was slight (<6%), found only in one generation, and seen only in the males, it was not considered to be adverse. The microscopic changes were consistent with an alpha-2u globulin-mediated process that is unique to male rats and not toxicologically relevant to humans.

 

Based on the data reported, the reproductive NOAEL as defined by this study is >20000 mg/m3.